Chimeric alkaline phosphatase-like proteins

ABSTRACT

The invention relates to improved alkaline phosphatases, pharmaceutical compositions comprising improved alkaline phosphatases and the use of improved alkaline phosphatases for preventing, treating or curing diseases.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

This application includes a Sequence Listing submitted electronicallyvia EFS-Web (name: “3151.0100003_Sequence_Listing.txt”; size: 21,688bytes; and created on: Oct. 16, 2020), which is hereby incorporated byreference in its entirety.

The invention relates to alkaline phosphatases with improved properties,pharmaceutical compositions comprising alkaline phosphatases withimproved properties and the use of alkaline phosphatases with improvedproperties for preventing, treating or curing diseases.

A phosphatase is an enzyme that dephosphorylates its substrates; i.e. ithydrolyses phosphoric acid monoesters into a phosphate ion and amolecule with a free hydroxyl group. This action is directly opposite tothat of phosphorylases and kinases, which attach phosphate groups totheir substrates by using energetic molecules like ATP. Phosphatases canbe categorized into two main categories: Cysteine-Dependent Phosphatases(CDPs) and metallo-phosphatases.

Metallo-phosphatases typically co-ordinate 2 catalytically essentialmetal ion(s) within their active site. There is currently some confusionabout the identity of these metal ions, as successive attempts toidentify them yield different answers. There is currently evidence thatthese metals could be magnesium, manganese, iron, zinc, or anycombination thereof. It is thought that a hydroxyl ion bridging the twometal ions takes part in nucleophilic attack on the phosphate group.

Phosphatases act in opposition to kinases/phosphorylases, which addphosphate groups to proteins. The addition or removal of a phosphategroup may activate or de-activate an enzyme (e.g., kinase signalingpathways) or enable a protein-protein interaction to occur (e.g., SH3domains); therefore phosphatases are integral to many signaltransduction pathways. It should be noted that phosphate addition andremoval do not necessarily correspond to enzyme activation orinhibition, and that several enzymes have separate phosphorylation sitesfor activating or inhibiting functional regulation. CDK, for example,can be either activated or deactivated depending on the specific aminoacid residue being phosphorylated. Phosphates are important in signaltransduction because they regulate the proteins to which they areattached. To reverse the regulatory effect, the phosphate is removed.This occurs on its own by hydrolysis, or is mediated by proteinphosphatases.

One type of phosphatase, alkaline phosphatase (ALP or AP) (EC 3.1.3.1),is a hydrolase enzyme responsible for removing phosphate groups frommany types of molecules, including, e.g., nucleotides, proteins, andalkaloids. Up to recently, the alkaline phosphatases were thought to bemost effective in an alkaline environment, as the name suggests.

It is thought in the art that one possible physiological role of AP maybe that it interacts with inflammatory molecules. First it had beenpostulated that AP dephosphorylates endotoxins and as such reduces theinflammatory response to these highly inflammatory molecules. Sincethen, other mechanisms of action have been postulated and pursued.However, up to now, despite its beneficial role in a multitude ofinflammatory and other diseases, the mechanism of action has not yetbeen fully elucidated. In the past, alkaline phosphatase of bovineorigin has been used in animal models and clinical trials for thetreatment of, for example, sepsis, acute kidney injury, inflammatorybowel disease, enterocolitis, ischemia reperfusion damage, or otherinflammatory diseases (Riggle et al, J Surg Res. 2013 March;180(1):21-6; Peters et al, J Pharmacol Exp Ther. 2013 January;344(1):2-7; Martinez-Moya et al, Pharmacol Res. 2012 August;66(2):144-53; Pickkers et al, Crit Care. 2012 Jan. 23; 16(1); Ramasamyet al, Inflamm Bowel Dis. 2011 February; 17(2):532-42; Lukas et al,Inflamm Bowel Dis. 2010 July; 16(7):1180-6; Bol-Schoenmakers et al, EurJ Pharmacol. 2010 May 10; 633(1-3):71-7; Heemskerk et al, Crit Care Med.2009 February; 37(2):417-23; Tuin et al, Gut. 2009 March; 58(3):379-87;Su et al, Crit Care Med. 2006 August; 34(8):2182-7; van Veen et al, Br JSurg. 2006 April; 93(4):448-56; van Veen, Infect Immun. 2005 July;73(7):4309-14; Verweij et al, Shock. 2004 August; 22(2):174-9).

Although at present, alkaline phosphatases, isolated from naturalsources as well as recombinantly engineered, are available that areuseful in both diagnostics and disease treatment, there is a need foralternative phosphatases with for example an altered (for exampleimproved) specific activity, stability (for example in vivo T_(1/2), orstability in respect of storage (shelf-life)) or substrate specificity.

The present invention provides such modified phosphatases, which haveimproved properties compared to a chimeric recombinant alkalinephosphatase that is extensively described in WO2008/133511.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides an isolated protein havingphosphatase activity, wherein said protein comprises an amino acidsequence of at least 200 consecutive amino acids having at least 90%sequence identity with SEQ ID NO: 5, an amino acid sequence of at least50 consecutive amino acids having at least 90% sequence identity withSEQ ID NO: 6, and an amino acid sequence of at least 40 consecutiveamino acids having at least 90% sequence identity with SEQ ID NO: 7,wherein the full length protein comprises an amino acid sequence havingat least 90% sequence identity with the full length amino acid sequenceof SEQ ID NO: 1, with the proviso that the amino acid at position 279 isleucine (L), the amino acid at position 328 is valine (V) and the aminoacid at position 478 is leucine (L).

In a preferred embodiment, the full length protein comprises an aminoacid sequence having at least 90%, at least 95%, or at least 98%sequence identity with the full length amino acid sequence of SEQ ID NO:1, with the proviso that the amino acid corresponding to position 279 isleucine (L), the amino acid corresponding to position 328 is valine (V)and the amino acid corresponding to position 478 is leucine (L).

With the term “corresponding to” here, is meant the particular position,relative to the N-terminal amino acid of the mature protein, which isdesignated “position 1”. The term “corresponding to” explicitly does notrefer to the particular amino acid residue on that particular position.

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and may be used interchangeably.

As used herein, where “amino acid sequence” is recited it refers to anamino acid sequence of a protein or peptide molecule. An “amino acidsequence” can be deduced from the nucleic acid sequence encoding theprotein. However, terms such as “polypeptide” or “protein” are not meantto limit the amino acid sequence to the deduced amino acid sequence, butcan include posttranslational modifications of the deduced amino acidsequences, such as amino acid deletions, additions, and modificationssuch as glycosylations and addition of lipid moieties. Also the use ofnon-natural amino acids, such as D-amino acids to improve stability orpharmacokinetic behaviour falls within the scope of the term “amino acidsequence”, unless indicated otherwise.

Preferably, part of said protein comprises an amino acid sequence of50-65, preferably 60-65, more preferably 62-65, more preferably 64-65,most preferably 65 consecutive amino acids, having at least 90%,preferably at least 95%, more preferably at least 98% sequence identitywith the full length crown domain of human PLAP. For the purpose of thepresent invention, the amino acid sequence corresponding to the fulllength crown domain of the human PLAP reference sequence is underlinedin SEQ ID NO: 3, depicted in FIG. 1 and corresponds to positions 366-430therein.

Preferably, part of said protein comprises an amino acid sequence of200-365, more preferably 250-365, more preferably 300-365, morepreferably 350-365, more preferably at least 360-365, most preferably365 consecutive amino acids having 90%, preferably at least 95%, morepreferably at least 98% sequence identity with the N-terminal regionflanking the crown domain of human ALPI. This N-terminal region flankingthe crown domain is considered one of two parts of the catalytic domain.

Preferably, part of said protein comprises an amino acid sequence of40-54, preferably 45-54, more preferably 50-54, more preferably 52-54,most preferably 54 consecutive amino acids having 90%, preferably atleast 95%, more preferably at least 98% sequence identity with theC-terminal region flanking the crown domain of human ALPI. ThisC-terminal region flanking the crown domain is considered the second oftwo parts of the catalytic domain.

For the purpose of the present invention, the amino acid sequence of thehuman ALPI mature protein reference sequence is depicted in FIG. 1 (SEQID NO: 2). For the purpose of determining the N-terminal and C-terminalflanking regions, together referred to as the catalytic domain, thecrown domain of human ALPI is herein underlined.

In a particular preferred embodiment, the invention provides a proteinaccording to the invention, wherein said protein comprises an amino acidsequence of 50-65, preferably 60-65, more preferably 62-65, morepreferably 64-65, most preferably 65 consecutive amino acids, having atleast 90%, preferably at least 95%, more preferably at least 98%sequence identity with the full length crown domain of human PLAP, partof said protein comprises an amino acid sequence of 200-365, morepreferably 250-365, more preferably 300-365, more preferably 350-365,more preferably 360-365, most preferably 365 consecutive amino acidshaving at least 90%, preferably at least 95%, more preferably at least98% sequence identity with the N-terminal region flanking the crowndomain of human ALPI, and part of said protein comprises an amino acidsequence of 40-54, preferably 45-54, more preferably 50-54, morepreferably 52-54, most preferably 54 consecutive amino acids having atleast 90%, preferably at least 95%, more preferably at least 98%sequence identity with the C-terminal region flanking the crown domainof human ALPI.

With N-terminal flanking region of the crown domain is meant a stretchof amino acids adjacent (i.e. preferably less than 20 amino acids, morepreferably less than 15, more preferably less than 10, more preferablyless than 5, more preferably less than 3, more preferably less than 2,most preferably no amino acid apart) to the sequence of the crown domain(the corresponding amino acids thereof underlined in FIG. 1), at theleft hand side of the crown domain, wherein the left hand side isdefined as that part of the peptide chain carrying the amino (NH2) groupof the first amino acid. With C-terminal flanking region of the crowndomain is meant a stretch of amino acids corresponding to the positionsadjacent (i.e. less than 20 amino acids, preferably less than 15, morepreferably less than 10, more preferably less than 5, more preferablyless than 3, more preferably less than 2, most preferably no amino acidapart) to the sequence of the crown domain (the corresponding aminoacids thereof underlined in FIG. 1), at right hand side of the crowndomain, wherein the right hand side is defined as that part of thepeptide chain carrying the free alpha carboxyl group of the last aminoacid.

In humans, four alkaline phosphatase isoforms have been identified sofar. These are intestinal (ALPI), placental (ALPP), placental-like(GCAP), and liver/bone/kidney (or tissue non-specific) alkalinephosphatase (TNAP). The first three are located together on chromosome 2while the tissue non-specific form is located on chromosome 1. The exactphysiological functions of the APs are not known, but AP appears to beinvolved in a large number of physiological processes.

The sequence of human alkaline phosphatases is known in the art and canbe easily found in the relevant databases. For determining the %sequence identity to the crown domain of PLAP and catalytic domain ofALPI, the respective reference sequences are preferably used. Thereference sequence of human ALPI is depicted as SEQ ID NO: 2. Thereference sequence of human ALPP is depicted as SEQ ID NO: 3. Withinthose reference sequences in FIG. 1, the sequence commonly known as thecrown domain is underlined.

The placental alkaline phosphatase is herein abbreviated as ALPP orPLAP. The abbreviations ALPI or IAP refer to intestinal alkalinephosphatase. The placental-like 2 alkaline phosphatase is hereinabbreviated as ALPP2, ALPG or GCAP and the abbreviations ALPL, TNSALP,TNAP or BLK are herein used to refer to liver/tissue non-specificalkaline phosphatase. The different abbreviations for one and the samealkaline phosphatase may be used interchangeably herein.

From a conformational point of view, an alkaline phosphatase roughlyconsists of two domains: a crown domain and an active-site domain(Mammalian Alkaline Phosphatases: From Biology to Applications inMedicine and Biotechnology. José Luis Millan; Wiley, 2006). Theactive-site domain can be divided in separate parts like the catalyticresidue and three metal ion sites (Zn1, Zn2 and Mg3). From a primarystructure point of view, it is clear that the crown domain is flanked bythe amino acids that form the active site domain. Hence, in a preferredembodiment, the catalytic domain is not composed of a contiguoussequence of amino acids, but is flanking the crown domain. Withreference to SEQ ID NO:1 which denotes the amino acid sequence of onealkaline phosphatase according to the invention, which does not limitthe present invention in any way, the crown domain preferably comprisesthe amino acids on position 366-430, whereas the catalytic domainpreferably refers to the remaining sequences before position 366 andafter position 430 in the mature protein sequences as depicted inFIG. 1. The amino acid sequence of alkaline phosphatases and therelative positions of the catalytic and crown domain are known by theskilled person (Mammalian Alkaline Phosphatases: From Biology toApplications in Medicine and Biotechnology. José Luis Millan; Wiley,2006).

In some embodiments, a protein according to the invention thus comprisesa sequence having at least 90% sequence identity with the crown domainof a human ALPP and a sequence having at least 90% sequence identitywith the catalytic domain of a human intestinal alkaline phosphatase.Preferably said sequence having said sequence identity to the crowndomain of ALPP is situated in a protein according to the invention atapproximately the same position as the crown domain of ALPP in thenative ALPP protein, i.e. at approximately position 366-430, relative toposition 1 as indicated in FIG. 1 (sequence representing the crowndomain is underlined).

The sequence having sequence identity with the crown domain of ALPPpreferably has at least 95%, more preferably at least 98%, mostpreferably 100% sequence identity with the native sequence of the crowndomain of ALPP, which is represented by underlined amino acids onpositions 366-430 in SEQ ID NO: 3.

The percentage of identity of an amino acid or nucleic acid sequence, orthe term “% sequence identity”, is defined herein as the percentage ofresidues in a candidate amino acid or nucleic acid sequence that isidentical with the residues in a reference sequence after aligning thetwo sequences and introducing gaps, if necessary, to achieve the maximumpercent identity. In a preferred embodiment, the calculation of said atleast percentage of sequence identity is carried out without introducinggaps. Methods and computer programs for the alignment are well known inthe art, for example “Align 2” or the BLAST service of the NationalCenter for Biotechnology Information (NCBI).

Preferably said sequence having a sequence which is at least 90%identical to the N-terminal part flanking the crown domain of ALPI issituated in a protein according to the application at approximately thesame position as that part of ALPI in the native ALPI protein, i.e. asrepresented by positions 1-365 in SEQ ID NO: 1 in FIG. 1.

Further, the sequence having sequence identity with the catalytic domainof ALPI preferably has at least 95%, more preferably at least 98%sequence identity with the N-terminal part flanking the crown domain ofALPI, represented by positions 1-365 in FIG. 1, SEQ ID NO: 2, with theproviso that the amino acid at position 279 is L and the amino acidcorresponding to position 328 is V. Most preferably, the sequence havingsequence identity with that N-terminal part of the catalytic domain ofALPI is identical to the native sequence of the catalytic domain ofALPI, with the exception that the amino acid at position 279 is L andthe amino acid at position 328 is V.

Preferably said sequence having a sequence which is at least 90%identical to the C-terminal part flanking the crown domain of ALPI issituated in a protein according to the invention at approximately thesame position as that part of ALPI in the native ALPI protein, i.e. asrepresented by positions 431-484 in SEQ ID NO: 1.

The sequence having sequence identity with the C-terminal part flankingthe crown domain of ALPI preferably has at least 95%, more preferably atleast 98% sequence identity with the C-terminal sequence, flanking thecrown domain of ALPI and represented by positions 431-484 in SEQ ID NO:2, with the proviso that the amino acid at position 478 is L. Mostpreferably, the sequence having sequence identity with that C-terminalpart of the catalytic domain of ALPI is identical to the native sequenceof the catalytic domain of ALPI, with the exception that the amino acidat position 478 is L.

Previously, it was shown that an alkaline phosphatase having a crowndomain sequence which has sequence identity with the crown domain ofALPP and having a catalytic domain having a sequence identity with thecatalytic domain of ALPI (herein referred to as catALPI/crownALPP)retained its initial specific activity in low Zn²⁺ medium. These resultsshowed that the in vivo activity is Zn²⁺ independent. In comparison,ALPI quickly lost its activity under the same low Zn²⁺ conditions. Theinventors concluded that such an enzyme whose activity is independent ofZn²⁺ could be useful in illnesses where Zn²⁺ depletion is part of thepathology (e.g. nutritional defects, alcohol abuse and intestinalintegrity damage, chronic infections including sepsis, or inflammatorydiseases in general) or where addition of Zn²⁺ (as a stabilizing agentin manufacture) may be contraindicated (e.g. acute phase of sepsis,autoimmune diseases). Apart from production and application advantages,catALPI/crownALPP also had advantages in respect to stability duringstorage. The properties of catALPI/crown ALPP are described in detail inWO2008/133511.

The current application surprisingly shows that a protein according tothe present invention is even more stable at low Zn²⁺ concentration. Itis shown here, that a specific modification involving three positionswith respect to the previously described catALPI/crownALPP sequenceincreases the Zn²⁺ independency even more. In summary, it has thus beenshown that native AP, such as ALPI, loses its enzymatic activity inenvironments with low Zn²⁺ concentrations, catALPI/crownALPP (asdescribed in WO2008/133511) retains its activity in environments withlow Zn²⁺ but loses its activity when, e.g. Zn²⁺ chelating agents areadded, whereas a protein according to the present invention retains muchof its activity even in the presence of a zinc chelator, such as EDTA.Thus, in diseases wherein severe Zn²⁺ depletion is part of thepathology, said native AP is unable to unfold its enzymatic activity atthe site where it is thought to be the most beneficial, e.g. at the siteof inflammation. In contrast, a recombinant AP not susceptible to verylow Zn²⁺ concentrations, in particular a protein according to theinvention retains its activity in an environment with very low Zn²⁺concentration, e.g. at an inflammation site. Such enzyme is thus veryuseful for treatment of diseases that are due to or accompanied by lowZn²⁺ levels. Such decreased Zn²⁺ levels would render other alkalinephosphatases less active, as compared to a protein according to theinvention.

Several enzymes in the human body depend on Zn²⁺ for their activity andfor instance immunologic responses are more effective if sufficientlevels of Zn²⁺ are present. The innate as well as the specific parts ofthe immune system are known to be influenced by zinc and it has beenestablished that zinc containing proteins accumulate at sites ofinflammation. In a healthy individual, Zn²⁺ serum reference values arebetween 10 and 20 μM. For instance in alcohol abuse or duringmalnutrition, these levels can decrease to less than 10 μM or even lessthan 1 μM. Furthermore, (sub)chronic inflammation, such as rheumatoidarthritis, sepsis, and Crohn's disease present with serum zincdeficiency. In such Zn²⁺ deficient environments, a protein according tothe invention is still very active whereas other, known alkalinephosphatases more or less quickly lose their phosphatase activity.

The invention thus provides the insight that a protein according to theinvention is especially useful in the treatment of a disease that isaccompanied with local or systemic Zn²⁺ deficiency. Compared to otherknown alkaline phosphatases, a protein according to the invention ismore active under low Zn²⁺ conditions. In preferred embodimenttherefore, the invention provides a protein according to the inventionfor use in preventing or treating a disease which is accompanied by Zn²⁺deficiency. Preferably, said disease comprises an inflammatory disease,more preferably selected from the group consisting of autoimmunediseases, rheumatoid arthritis, asthma, chronic obstructive pulmonarydisease, atherosclerosis, inflammatory disease of the gastro-intestinaltract, infection, sepsis, neurodermatitis, inflammatory liver disease,inflammatory lung disease and inflammatory kidney disease.

With the term “zinc deficiency” or “Zn²⁺ deficiency” is meant that theamount of zinc (locally) available is insufficient to enable unhinderedcellular and/or enzymatic activity. Zinc deficiency can be absolute orrelative, wherein absolute zinc deficiency can be easily determined byreferring to reference values in healthy individuals, known in the art.Relative zinc deficiency, for instance, can occur when zincconcentrations are still within the boundaries set by reference valuesin healthy individuals, but zinc concentrations needed are higher thannormal, for instance during inflammation. In some embodiments, zincdeficiency means that a subject's (local) zinc concentration is below 10μM, more preferably below 5 μM, more preferably below 2 μM, morepreferably below 1 μM, more preferably below 0.1 μM, more preferablybelow 0.05 μM, more preferably below 0.02 μM, more preferably below 0.01μM, more preferably below 0.005 μM, most preferably below 0.002 μM. Insome embodiments, zinc deficiency means that the (local) zincconcentration is lower than necessary for unhindered cellular and/orenzymatic activity.

With the term “inflammatory disease” is meant, any disease that is dueto or accompanied by a protective tissue response to injury ordestruction of tissues, which serves to destroy, dilute, or wall offboth the injurious agent and the injured tissues. The classical signs ofacute inflammation are pain (dolor), heat (calor), redness (rubor),swelling (tumor), and loss of function (functio laesa). Typically,inflammation is characterized by increase in inflammatory parameters,such as C-reactive protein, leucocytes and cytokines (IL-6, TNF-alpha,etc). Although the inflammation is initially protective by nature,deranged inflammatory disease, such as rheumatoid arthritis and (other)autoimmune diseases, also fall within the definition of “inflammatorydisease”. In a preferred embodiment, the protein of the presentinvention is for use in the treatment of such deranged or harmfulinflammatory disease.

Further, during standard pharmacokinetic analysis, the present inventorshave unexpectedly observed that a protein according to the invention istargeted to several organs, in particular skin, kidney, spleen, liver,lungs, brain, fat, bone, and colon. Using a radioactive iodine-coupledprotein of the invention, it has been observed that the % organ/bloodratio for a protein according to the invention relative to that ofcatALPI/crownALPP is especially favourable for skin, kidney, spleen,liver, lungs, brain, fat, bone, and colon, i.e. a protein according tothe invention is targeted relatively more to these organs than the knowncatALPI/crownALPP protein. In particular, a protein according to theinvention resides in lower concentrations in the blood thancatALPI/crownALPP and resides in higher concentrations in the mentionedorgans. When a condition relating to these organs, such as liverneurodermatitis, kidney injury, liver fibroses, hypophosphatasia or thelike is to be treated less protein is needed for treatment, reducingcosts and/or increasing efficacy. Experiments involving diseases relatedto these organs have already been performed (hypophosphatasia), are inprogress (kidney disease), or are planned. The invention thus provides aprotein which shows improved zinc independency and pharmacokineticbehaviour when compared to alkaline phosphatases known in the art.

The inventors have shown, in various working embodiments, that a proteinaccording to the invention is useful as a medicament. Diseases orconditions that can be treated with a protein according to the inventioninclude: reduced renal function, kidney injury, renal failure andhypophosphatasia. Further, because of specific features of an alkalinephosphatase protein according to the invention which constituteimprovements over known alkaline phosphatase proteins that have beenused, the protein according to the invention is useful not only forreduced renal function, kidney injury, renal failure andhypophosphatasia, but also for the prevention or treatment of autoimmunediseases, rheumatoid arthritis, asthma, chronic obstructive pulmonarydisease, atherosclerosis, inflammatory disease of the gastro-intestinaltract, infection, sepsis, neurodermatitis, inflammatory liver disease,inflammatory lung disease and inflammatory kidney disease. These are alldiseases affecting organs which are targeted by a protein according tothe invention and/or are accompanied by (relative) zinc deficiency.

The term “prevention” or “preventing” is herein defined as the (the actof) administering a protein according to the invention to a subject withthe intention to prevent said subject of attracting a particulardisease. Although the intention of treatment is to prevent said subjectattracting said disease, it is not necessary that the disease state iscompletely prevented after one or multiple administration(s) of aprotein according to the invention, as subjects are, e.g., notnecessarily susceptible for a specific treatment protocol. It ispreferred that, overall, efficacy of said prevention is achieved in thata group of subjects that has received a protein according to theinvention for preventing a particular disease shows at least a reducedincrease in disease-severity or e.g. less complications of said disease,when compared with a group of subjects that has not received saidprotein. For instance in case of renal disease, it is preferred thatsubjects at risk of attracting renal disease, after being treated with aprotein according to the invention show less decline in renal function,relative to subjects that have not been treated with said protein. If asubject attracts a disease for which a protein was administered in orderto prevent said disease, further administrations may either be given(herein referred to as “treatment”) to prevent worsening of said diseasestates or as treatment with the intention to cure.

The term “treatment” or “treating” is herein defined as (the act of)administering a protein according to the invention to a subject with theintention to cure the subject of an (expected) illness or improve,reduce, or remove the symptoms of an illness in the subject. Althoughthe intention of treatment is to cure said subject, it is not necessarythat said subject is cured after one or multiple administration(s) of aprotein according to the invention, as subjects are, e.g., notnecessarily susceptible for a specific treatment protocol. It ispreferred that, overall, efficacy of said treatment is achieved in thatsubjects having been treated with a protein according to the inventionshow at least an improvement of their disease-condition or e.g. lesscomplications of said disease, when compared with a non-treated (orplacebo treated) group. For instance in case of renal disease, it ispreferred that subjects, after being treated with a protein according tothe invention show improved renal function or less decline in renalfunction, relative to subjects that have not been treated with saidprotein.

The invention further provides a polynucleotide comprising a nucleotidesequence encoding a protein according to the invention.

As used herein, the terms “nucleic acid sequence” and “polynucleotide”also encompass non-natural molecules based on and/or derived fromnucleic acid sequences, such as for instance artificially modifiednucleic acid sequences, peptide nucleic acids, as well as nucleic acidsequences comprising at least one modified nucleotide and/or non-naturalnucleotide such as for instance inosine, LNA, Morpholino, and2′-O-methyl RNA.

Also provided is a vector comprising such polynucleotide according tothe invention. Such a vector preferably comprises additional nucleicacid sequences such as elements necessary for transcription/translationof the nucleic acid sequence encoding a phosphatase (for examplepromoter and/or terminator sequences). Said vector can also comprisenucleic acid sequences coding for selection markers (for example anantibiotic) to select or maintain host cells transformed with saidvector. Examples of suitable vectors are cloning or expression vectors.Any vector suitable for mediating expression in a host cell of choicemay be used according to the invention, either integrated or episomallyreplicating in a host cell. The vector can be a plasmid, a virus (forexample a retrovirus, adenovirus, adeno-associated virus, baculovirusand/or derivatives thereof), a cosmid, a phage or a phagemid, anepisomal vector or an artificial chromosome. Such polynucleotide orvector is very useful for the production of a protein according to theinvention, but can also be used for gene therapy.

The invention thus provides a protein according to the invention for useas a medicament. It is also possible to treat a patient suffering fromor at risk of suffering from any one of the above mentioned disease tobe treated with a polynucleotide according to the invention, or with avector according to the invention, in order to express a proteinaccording to the invention in vivo. The invention thus also provides apolynucleotide according to the invention or a vector according to theinvention for use as a medicament, preferably for the prevention ortreatment of reduced renal function, kidney injury, renal failure,hypophosphatasia autoimmune diseases, rheumatoid arthritis, asthma,chronic obstructive pulmonary disease, atherosclerosis, inflammatorydisease of the gastro-intestinal tract, infection, sepsis,neurodermatitis, inflammatory liver disease, inflammatory lung diseaseand inflammatory kidney disease.

Also provided is a protein, a polynucleotide or a vector according tothe invention for use in a method for the prevention or treatment of aninflammatory disease, a kidney disease or hypophosphatasia.

Also provided is the use of a protein, polynucleotide and/or vector foraccording to the invention for the manufacturing of a medicament,preferably for the prevention or treatment of reduced renal function,kidney injury, renal failure, hypophosphatasia, autoimmune diseases,rheumatoid arthritis, asthma, chronic obstructive pulmonary disease,atherosclerosis, inflammatory disease of the gastro-intestinal tract,infection, sepsis, neurodermatitis, inflammatory liver disease,inflammatory lung disease and inflammatory kidney disease.

In another embodiment, the invention provides the use of an alkalinephosphatase protein according to the invention, of a polynucleotideaccording to the invention, or of a vector according to the invention,in the preparation of a medicament for the treatment of a disease whichis accompanied by a local or systemic Zn²⁺ deficiency, preferably saiddisease is an inflammatory disease, a kidney disease orhypophosphatasia, preferably said disease comprises an inflammatorydisease, more preferably a disease selected from the group consisting ofautoimmune diseases, rheumatoid arthritis, asthma, chronic obstructivepulmonary disease, atherosclerosis, inflammatory disease of thegastro-intestinal tract, infection, sepsis, neurodermatitis,inflammatory liver disease, inflammatory lung disease and inflammatorykidney disease.

In yet another embodiment, the invention provides a method for treatinga subject (preferably a human) to treat a disease which is preferablyaccompanied by Zn²⁺ deficiency, comprising administering an effectiveamount of a phosphatase according to the invention, wherein said diseasepreferably comprises an inflammatory disease, more preferably selectedfrom the group consisting of autoimmune diseases, rheumatoid arthritis,asthma, chronic obstructive pulmonary disease, atherosclerosis,inflammatory disease of the gastro-intestinal tract, infection, sepsis,neurodermatitis, inflammatory liver disease, inflammatory lung diseaseand inflammatory kidney disease.

The invention thus provides a protein, polynucleotide, and/or vectoraccording to the invention for use in a variety of disease. A proteinaccording to the invention is especially useful, because of its organdistribution, for use in treating a disease involving thegastro-intestinal tract, kidney skin, liver, lung, brain, fat tissue, orbone.

In one preferred embodiment, the invention provides a protein,polynucleotide and/or vector according to the invention for use intreating kidney disease.

Although there is continuing insight in the pathophysiology of kidneyinjury and there are therapies that improve renal function [Lameire N H,Acute kidney injury: an increasing global concern. Lancet. 2013 Jul.13:382(9887):170-9], there is still a need for alternative treatments totreat kidney injury and/or improve renal function. The present inventionprovides such alternative treatment by providing a protein,polynucleotide and/or a vector for use according to the invention.

In a preferred embodiment, the invention provides a protein,polynucleotide and/or vector for use according to the invention, whereinsaid kidney disease is selected from the group of renal failure, acutekidney injury, chronic kidney disease, ischemic renal disease.

The term acute kidney injury (AKI), previously called acute renalfailure, means that kidney function is rapidly lost. AKI is diagnosed onthe basis of characteristic laboratory findings, such as elevated bloodurea nitrogen and creatinine, or inability of the kidneys to producesufficient amounts of urine. AKI can be subdivided in prerenal AKI,intrinsic AKI and postrenal AKI.

Prerenal AKI is caused by decreased effective blood flow to the kidney.Typical laboratory findings for prerenal AKI are: U_(osm):>500,U_(Na):<10, Fe_(Na):<1% and BUN/Cr ratio:>20.

The term intrinsic AKI is used when sources of damage to the kidneyitself are the cause. Typical laboratory findings for intrinsic AKI are:U_(osm): <350, U_(Na):>20, Fe_(Na):>2% and BUN/Cr ratio:<15.

The term postrenal AKI is reserved for those conditions were urinarytract obstruction is the cause of AKI. Typical laboratory findings forpostrenal AKI are: U_(osm):<350, U_(Na):>40, Fe_(Na):>4% and BUN/Crratio:>15.

Worldwide, there are several guidelines for classification of chronickidney disease. As one example, the classification criteria of theNational Kidney Foundation (2002) “K/DOQI clinical practice guidelinesfor chronic kidney disease” are described below. The skilled person mayselect different patient populations, based on other guidelines.

Typically, all individuals with a glomerular filtration rate (GFR)<60mL/min/1.73 m2 for 3 months are classified as having chronic kidneydisease, irrespective of the presence or absence of kidney damage. Therationale for including these individuals is that reduction in kidneyfunction to this level or lower represents loss of half or more of theadult level of normal kidney function, which may be associated with anumber of complications. Generally individuals with chronic kidneydamage are classified as having chronic kidney disease, irrespective ofthe level of GFR. The rationale for including individuals with GFR >60mL/min/1.73 m2 is that GFR may be sustained at normal or increasedlevels despite substantial kidney damage and that patients with kidneydamage are at increased risk of the two major outcomes of chronic kidneydisease: loss of kidney function and development of cardiovasculardisease, which may lead to morbidity and mortality.

The loss of protein in the urine is regarded as an independent markerfor worsening of renal function and cardiovascular disease. Hence,British guidelines append the letter “P” to the stage of chronic kidneydisease if there is significant protein loss.

The 5 stages of chronic kidney disease are typically classified asfollows:

Stage 1: Slightly diminished function; kidney damage with normal orrelatively high GFR (≥90 mL/min/1.73 m2). Kidney damage is defined aspathological abnormalities or markers of damage, including abnormalitiesin blood or urine test or imaging studies.

Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m2) with kidneydamage. Kidney damage is defined as pathological abnormalities ormarkers of damage, including abnormalities in blood or urine test orimaging studies.

Stage 3: Moderate reduction in GFR (30-59 mL/min/1.73 m2). Britishguidelines distinguish between stage 3A (GFR 45-59) and stage 3B (GFR30-44) for purposes of screening and referral.

Stage 4: Severe reduction in GFR (15-29 mL/min/1.73 m2) Preparation forrenal replacement therapy.

Stage 5: Established kidney failure (GFR <15 mL/min/1.73 m2), permanentrenal replacement therapy (RRT), or end stage renal disease (ESRD).

The term renal failure now denotes a medical condition in which thekidneys fail to adequately filter waste products from the blood. The twomain forms are acute kidney injury, which is often reversible withadequate treatment, and chronic kidney disease, which is often notreversible.

Ischemic renal disease is reserved for those conditions in which aclinically important reduction in glomerular filtration rate or loss ofrenal parenchyma is caused by hemodynamically significant renal arterystenosis or other causes that result in low blood pressure in thekidneys, such as e.g. hemodynamic shock or the use of an arterial clampfor temporary interruption of blood flow.

The inventors have also shown that a protein according to the inventionis useful in the treatment of hypophosphatasia (HPP). This was quiteunexpected, as previous experiments using TNAP for the treatment ofhypophosphatasia failed to show efficacy of enzyme replacement therapy.Consequently an artificial fusion protein between a TNAP enzyme and abone homing peptide was developed (Whyte et al, N Engl J Med. 2012 Mar.8; 366(10):904-13). Surprisingly, the protein according to the presentinvention does not need such bone homing peptide and is able toeffectively reduce the signs of symptoms of hypophosphatasia in a mousemodel. In another preferred embodiment, the invention thus provides aprotein, polynucleotide and/or vector according to the invention for usein treating hypophosphatasia.

The metabolic basis of hypophosphatasia stems from a molecular defect inthe gene encoding tissue non-specific alkaline phosphatase (TNAP). TNAPis an ectoenzyme tethered to the outer surface of osteoblast andchondrocyte cell membranes. TNAP normally hydrolyzes several substances,including inorganic pyrophosphate (PPi) and pyridoxal 5′-phosphate (PLP)a major form of vitamin B6.

When TNAP is low, inorganic pyrophosphate (PPi) accumulatesextracellularly and potently inhibits formation of hydroxyapatite(mineralization) causing rickets in infants and children andosteomalacia (soft bones) in adults. PLP is the principal form ofvitamin B6 and must be dephosphorylated by TNAP to pyridoxal (PL) tocross over the cell membrane. Vitamin B6 deficiency in the brain impairssynthesis of neurotransmitters, which can cause seizures. In some cases,deposition of calcium pyrophosphate dehydrate (CPPD) crystals in thejoint can cause pseudogout.

There are no approved therapies for HPP today. Current managementconsists of palliating symptoms, maintaining calcium balance andapplying physical, occupational, dental and orthopedic interventions asnecessary. Bisphosphonate (pyrophosphate synthetic analog)administration in one infant had no discernible effect on the skeleton,and the infant's disease progressed until death at 14 months of age.

Bone marrow cell transplantation in two severely affected infantsproduced radiographic and clinical improvement, although the mechanismof efficacy is not fully understood and significant morbidity remained.Enzyme replacement therapy with normal serum or with ALP-rich serum frompatients with Paget's bone disease was not beneficial [Whyte M P, ValdesR, Ryan L M, McAlister W H (September 1982). “Infantilehypophosphatasia: enzyme replacement therapy by intravenous infusion ofalkaline phosphatase-rich plasma from patients with Paget bone disease”.J. Pediatr. 101 (3): 379-86] [Whyte M P, McAlister W H, Patton L S, etal. (December 1984). “Enzyme replacement therapy for infantilehypophosphatasia attempted by intravenous infusions of alkalinephosphatase-rich Paget plasma: results in three additional patients”. J.Pediatr. 105 (6): 926-33]. These enzyme replacement therapies werethought to be ineffective because the alkaline phosphatase function issupposed to be at the bone surface and not in the blood. Furtherattempts to improve the efficacy of enzyme replacement therapy weretherefore built around a bone targeted TNAP enzyme with promisingresults [Whyte et al. N Engl J Med. 2012 Mar. 8; 366(10):904-13].Surprisingly, the present invention shows that, without introducing anartificial bone-targeting moiety, a protein according to the inventionis beneficial in a murine model of hypophosphatasia. One advantage ofthe present protein over that described in Whyte et al and Millan et al[J. Bone Miner. Res. 2008; 23(6):777-787] is that, because of theabsence of the artificial bone targeting moiety, the present protein isexpected to be much less immunogenic. The present protein consists of aprotein having high sequence identity with naturally occurring humanalkaline phosphatase proteins. Furthermore, the protein of the presentinvention has an advantage over the bone-targeted alkaline phosphatasewith respect to ease and cost of production.

In a preferred embodiment, a protein, polynucleotide, and/or vectoraccording to the invention for use in the treatment of hypophosphatasiais provided, wherein said hypophosphatasia is selected from perinatalhypophosphatasia, infantile hypophosphatasia, childhoodhypophosphatasia, and adult hypophosphatasia.

Perinatal hypophosphatasia is the most pernicious form ofhypophosphatasia. In utero, profound hypomineralization results in caputmembraneceum, deformed or shortened limbs during gestation and at birthand rapid death due to respiratory failure.

Infantile hypophosphatasia presents in the first 6 months of life.Postnatal development often appears normal until the onset of poorfeeding and inadequate weight gain, and clinical manifestations ofrickets are recognized. Hypercalcemia and hypercalcinuria are alsocommon and may explain the nephrocalcinosis, renal compromise, andepisodes of recurrent vomiting. Mortality is estimated to be 50% in thefirst year of life.

Hypophosphatasia in childhood has variable clinical expression. As aresult of aplasia, hypoplasia, or dysplasia of dental cementum,premature loss of deciduous teeth (i.e. before the age of 5) occurs.Frequently, incisors are shed first; occasionally almost the entireprimary dentition is exfoliated prematurely. Dental radiographssometimes show the enlarged pulp chambers and root canals characteristicof the “shell teeth” of rickets. Patients may also experience delayedwalking, a characteristic waddling gait, complain of stiffness and pain,and have an appendicular muscle weakness (especially in the thighs)consistent with non-progressive myopathy. Typically, radiographs showrachitic deformities and characteristic bony defects near the ends ofmajor long bones (i.e. “tongues” of radiolucency projecting from therachitic growth plate into the metaphysis). Growth retardation, frequentfractures and osteopenia are common. In severely affected infants andyoung children it is not uncommon, despite the appearance of widely“open” fontanels on radiographic studies, for functional synostosis ofcranial sutures to occur. The illusion of “open” fontanels results fromlarge areas of hypomineralized calvarium. Subsequently true prematurebony fusion of cranial sutures may elevate intracranial pressure.

Adult hypophosphatasia can be associated with rickets, premature loss ofdeciduous teeth, or early loss of adult dentition followed by relativelygood health. Osteomalacia manifests in painful feet resulting fromrecurrent poorly healing metatarsal stress fractures, and discomfort inthe thighs or hips due to femoral pseudofractures which, when theyappear in radiographic study, are distinguished from most other types ofosteomalacia (which occur medially) by their location in the lateralcortices of the proximal femora. Some patients suffer from calciumpyrophosphate dihydrate crystal depositions with occasional overtattacks of arthritis (pseudogout), which appears to be the result ofelevated endogenous inorganic pyrophosphate (PPi) levels. These patientsmay also suffer articular cartilage degeneration and pyrophosphatearthropathy. Radiographs may reveal pseudofractures in the lateralcortices of the proximal femora, stress fractures, and patients mayexperience osteopenia, chondrocalcinosis, features of pyrophosphatearthropathy, and calcific periarthritis.

In a preferred embodiment, a protein, polynucleotide, and/or vector foruse in the treatment of hypophosphatasia according to the invention isprovided, wherein the treatment results in prolonged survival; increasedbody weight; improved skeletal phenotype (such as induction of secondaryossification centres, improvement of bone mineralization, for instancein trabecular and/or cortical bone, or induction of osteoid);attenuation of craniofacial defects (such as shape abnormalities andcoronal suture fusion); improvement of dento-alveolar phenotype (such asimprovement of molar height and form, dentin-thickness, and bonemineralization); and/or reduction of plasma PPi levels of the patient.

Also provided is the use of a protein, polynucleotide, and/or vectoraccording to the invention for the preparation of a medicament for theprevention or treatment of hypophosphatasia, wherein the treatmentresults in prolonged survival; increased body weight; improved skeletalphenotype (such as induction of secondary ossification centres,improvement of bone mineralization, for instance in trabecular and/orcortical bone, or induction of osteoid); attenuation of craniofacialdefects (such as shape abnormalities and coronal suture fusion);improvement of dento-alveolar phenotype (such as improvement of molarheight and form, dentin-thickness, and bone mineralization); and/orreduction of plasma PPi levels of a patient.

Also provided is a method for treating a subject suffering or at risk ofsuffering from hypophosphatasia, wherein the treatment results inprolonged survival; increased body weight; improved skeletal phenotype(such as induction of secondary ossification centres, improvement ofbone mineralization, for instance in trabecular and/or cortical bone, orinduction of osteoid); attenuation of craniofacial defects (such asshape abnormalities and coronal suture fusion); improvement ofdento-alveolar phenotype (such as improvement of molar height and form,dentin-thickness, and bone mineralization); and/or reduction of plasmaPPi levels in said subject.

In a more preferred embodiment, a protein, polynucleotide, and/or vectorfor use in the treatment of hypophosphatasia according to the inventionis provided, wherein said hypophosphatasia is selected from infantile,childhood or adult hypophosphatasia. More preferably, thehypophosphatasia is infantile hypophosphatasia.

Throughout the specification, examples and literature in the art, othernomenclature is used to designate the respective isoforms of alkalinephosphatase. For the sake of clarity, in Table 1 below the names andabbreviations commonly used, or used in this application are listed.

TABLE 1 synonyms and abbreviations used in the present patentapplication or generally known for the different types of alkalinephosphatases ALKALINE PHOSPHATASES ABBREVIATIONS Placental alkalinephosphatase ALPP, PLAP, Secretable Placental alkaline phosphataseshPLAP, sALPP Intestinal alkaline phosphatase ALPI, IAP hIAP SecretableIntestinal alkaline phosphatase shIAP, sALPI Placental-like alkalinephosphatase GCAP Tissue nonspecific alkaline phosphatase TNAP, BLK,ALPL, TNSALP Recombinant alkaline phosphatase catALPI/crownALPP,comprising the catalytic domain of ALPI RecAP, Xinplap, sALPI- and thecrown domain of ALPP ALPP-CD Protein according to the inventionLVL-RecAP, improved RecAP

With the term “secretable” is meant that no posttranslationalGlycosylphosphatidylinositol (GPI) anchor has been attached to matureprotein, enabling the protein to be secreted and not membrane bound. AGPI anchor is a glycolipid that can be attached to the C-terminus of aprotein during posttranslational modification. It is composed of aphosphatidylinositol group linked through a carbohydrate-containinglinker (glucosamine and mannose to the inositol residue through aglycoside bond) and via an ethanolamine phosphate (EtNP) bridge to theC-terminal amino acid of a mature protein. The two fatty acids withinthe hydrophobic phosphatidylinositol group anchor the protein to thecell membrane. It is advantageous for production and downstreamprocessing, and thus preferred that a protein according to the inventiondoes not comprise a GPI anchor.

It is clear that any of the described secretable modified phosphatases(and thus also a secretable protein according to the invention) can forexample be produced by introducing into a host cell a nucleic acidcapable of encoding said secretable phosphatase in operable linkage withregulatory sequences and allowing said host cell to express saidsecretable phosphatase and optionally isolating the produced phosphatasefrom the medium in which the host cell are grown and/or maintained.However, apart from mutations in the above-mentioned GPI-attachmentsequence, other methods exist that make GPI-anchorless, secretedproteins, e.g.:

-   -   1) After expression as membrane-anchored proteins,        phospholipases may be used to cleave off the GPI anchor. Hence        the invention also provides a method for producing a secreted        phosphatase comprising culturing a host capable of expressing a        membrane anchored phosphatase, allowing said host cell to        produce said phosphatase and incubating the obtained cells with        a phospholipase and optionally isolating the released        phosphatase.    -   2) Interference with the production of the GPI anchor or the use        of a cell (type) that is deficient in GPI anchor production may        also be used to make a secretable form of an otherwise        GPI-anchored protein. Examples of cell lines that have been made        to be deficient in GPI anchoring biochemistry are e.g. Jurkat,        AM-B, C84, BW, S49, CHO and Raji. In yet another embodiment the        invention therefore provides a method for producing a secreted        phosphatase comprising culturing a host cell capable of        expressing a secretable (alkaline) phosphatase (for example a        host cell comprising a nucleic acid sequence encoding any of the        mentioned modified secreted (alkaline) phosphatases), allowing        said host to produce said secretable phosphatase and optionally        isolating the produced phosphatase, wherein said host cell is        not capable of biosynthesis of functional GPI anchored proteins.        However, the host cell may also produce a phosphatase with a        functional GPI signal sequence.    -   3) Interference with or the use of a cell deficient in        transamidases may be used to inhibit attachment of a GPI anchor        to the protein, rendering the protein anchorless and secretable.        Such a deficient cell has been obtained through mutagenesis in        CHO.

A vector according to the invention preferably comprises additionalnucleic acid sequences such as elements necessary fortranscription/translation of the nucleic acid sequence encoding aphosphatase (for example promoter and/or terminator sequences). Saidvector can also comprise nucleic acid sequences coding for selectionmarkers (for example an antibiotic) to select or maintain host cellstransformed with said vector. Preferably, a nucleotide sequence encodingfor a GPI anchor sequence is absent in a polynucleotide and/or vectoraccording to the invention. Examples of suitable vectors are cloning orexpression vectors. Any vector suitable for mediating expression in asuitable host cell may be used according to the invention, eitherintegrated or episomally replicating in a host cell. The vector can be aplasmid, a virus (for example a retrovirus, adenovirus, adeno-associatedvirus, baculovirus or derivatives thereof), a cosmid, a phage or aphagemid, an episomal vector or an artificial chromosome.

Furthermore the invention also provides a host cell comprising aprotein, a nucleic acid sequence or vector according to the invention asdescribed. The cell can be a eukaryotic cell, preferably a mammaliancell, a plant cell or a yeast cell, that is suitable for production ofrecombinant proteins. Suitable yeast host cells comprise, e.g.,Saccharomyces cerevisiae and Pichia pastoris. Preferred host cells aremammalian (or more preferred human) derived cells such as BHK, HEK293,CHO or PerC6™. In a particular preferred embodiment, the host cell is aCHO cell.

A nucleic acid sequence encoding a protein according to the invention, avector comprising said nucleic acid sequence, and/or a host cellcomprising said nucleic acid sequence are very useful in the productionof a protein according to the invention. A protein according to theinvention comprises glycosylation sites and hence the protein ispreferably produced in cells that provide the desired glycosylationpattern. In a preferred embodiment, the used production system is amammalian (for example human) in vitro production platform and even morepreferably the production involves large-scale production. In anotherpreferred embodiment, the used production system is a plant or yeast ormammalian (preferably non-human) platform in which an artificialhuman-like glycosylation pattern is introduced.

In one embodiment, the invention thus provides a method for producing aprotein according to the invention, the method comprising culturing ahost cell comprising a polynucleotide according to the invention or avector according to the invention and allowing the host cell to producesaid protein. Preferred host cells are mammalian (or more preferredhuman) derived cells such as BHK, HEK293, CHO or PerC6™. In a particularpreferred embodiment, the host cell is a CHO cell. Preferably, a methodfor producing a protein according to the invention further comprisesharvesting and optionally purifying said protein from said culture.

As already mentioned, a herein described protein according to theinvention is useful in therapy. In one embodiment, the inventionprovides a composition, preferably a pharmaceutical composition,comprising a protein according to the invention. Said pharmaceuticalcomposition optionally comprises a pharmaceutical acceptable carrier,diluent or excipient.

The composition can be presented in any form, for example as a tablet,as an injectable fluid or as an infusion fluid etc. Moreover, thecomposition, protein, nucleotide and/or vector according to theinvention can be administered via different routes, for exampleintravenously, rectally, bronchially, or orally. Yet another suitableroute of administration is the use of a duodenal drip.

In a preferred embodiment, the used route of administration is theintravenous route. It is clear for the skilled person, that preferablyan effective amount of a protein according to the invention isdelivered. As a start point, 1-50,000 U/kg/day can be used. Anothersuitable route, e.g., for HPP, is the subcutaneous route. If theintravenous route of administration is used, a protein according to theinvention can be (at least for a certain amount of time) applied viacontinuous infusion.

Said composition according to the invention can optionally comprisepharmaceutically acceptable excipients, stabilizers, activators,carriers, permeators, propellants, desinfectants, diluents andpreservatives. Suitable excipients are commonly known in the art ofpharmaceutical formulation and may be readily found and applied by theskilled artisan, references for instance Remmington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia Pa., 17th ed. 1985.

For oral administration, the protein can, for example, be administeredin solid dosage forms, such as capsules, tablets (e.g., with an entericcoating), and powders, or in liquid dosage forms, such as elixirs,syrups, and suspensions. AP can be encapsulated in gelatine capsulestogether with inactive ingredients and powdered carriers, such asglucose, lactose, sucrose, mannitol, starch, cellulose or cellulosederivatives, magnesium stearate, stearic acid, sodium saccharin, talcum,magnesium carbonate and the like. Examples of additional inactiveingredients that can be added to provide desirable colour, taste,stability, buffering capacity, dispersion or other known desirablefeatures are red iron oxide, silica gel, sodium lauryl sulphate,titanium dioxide, edible white ink and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain colouring and flavouring to increasepatient acceptance.

In a preferred embodiment, a composition comprising a protein accordingto the invention is suitable for oral administration and comprises anenteric coating to protect the AP from the adverse effects of gastricjuices and low pH. Enteric coating and controlled release formulationsare well known in the art. Enteric coating compositions in the art maycomprise of a solution of a water-soluble enteric coating polymer mixedwith the active ingredient(s) and other excipients, which are dispersedin an aqueous solution and which may subsequently be dried and/orpelleted. The enteric coating formed offers resistance to attack of aprotein according to the invention by atmospheric moisture and oxygenduring storage and by gastric fluids and low pH after ingestion, whilebeing readily broken down under the alkaline conditions which exist inthe lower intestinal tract.

The invention provides a composition according to the invention for useas a medicament, preferably for treating a disease accompanied by alocal or systemic zinc deficiency, an inflammatory diseases, a kidneydisease, or hypophosphatasia. The inflammatory disease preferably beingselected from the group consisting of autoimmune diseases, rheumatoidarthritis, asthma, chronic obstructive pulmonary disease,atherosclerosis, inflammatory disease of the gastro-intestinal tract,infection, sepsis, neurodermatitis, inflammatory liver disease,inflammatory lung disease and inflammatory kidney disease. The kidneydisease preferably being selected from the group consisting of renalfailure, acute kidney injury, chronic kidney disease, and ischemic renaldisease. The hypophosphatasia preferably being selected from the groupconsisting of perinatal hypophosphatasia, infantile hypophosphatasia,childhood hypophosphatasia, and adult hypophosphatasia.

Besides the fact that a protein according to the invention can beincorporated in a pharmaceutical composition such a phosphatase can alsobe part of a nutritional composition or a nutraceutical.

A protein according to the invention can be added to a nutrient (such asmilk) but can also be produced within said nutrient (for instance bymolecular engineering). Moreover, tablets and/or capsules can beprepared which are subsequently added to a nutrient or which can betaken directly by a human being.

Further provided is a method for treating a subject suffering from aninflammatory disease, a kidney disease or hypophosphatasia, comprisingadministering an effective amount of a protein according to theapplication, e.g., an isolated or recombinant protein having phosphataseactivity, wherein part of said protein comprises an amino acid sequenceof at least 50 consecutive amino acids, having at least 90% sequenceidentity with the full length crown domain of human PLAP, part of saidprotein comprises an amino acid sequence of at least 200 consecutiveamino acids having 90% sequence identity with the N-terminal regionflanking the crown domain of human ALPI, and part of said proteincomprises an amino acid sequence of at least 40 consecutive amino acidshaving 90% sequence identity with the C-terminal region flanking thecrown domain of human ALPI., wherein the full length protein comprisesan amino acid sequence having at least 90% sequence identity with thefull length amino acid sequence of SEQ ID NO: 1, with the proviso thatthe amino acid on position 279 is leucine (L), the amino acid onposition 328 is valine (V) and the amino acid on position 478 is leucine(L), or an effective amount of a polynucleotide according to theapplication or an effective amount of a vector according to theapplication.

The invention will be explained in more detail in the following,non-limiting examples.

FIGURE LEGENDS

FIG. 1: Amino acid sequences of mature protein LVL-RecAP, hIAP andhPLAP. The putative crown domains of the different proteins areunderlined.

FIG. 2: Organ distribution of LVL-RecAP compared to catALPI/crownALPP.Depicted is the organ to blood distribution of LVL-RecAP divided by theorgan to blood distribution of catALPI/crownALPP (=ratio of y-axis). Ahigher value is indicative of LVL-RecAP targeting the respective organwhen compared to catALPI/crownALPP.

FIG. 3: Survival of Akp2^(−/−) mice treated with either vehicle, 1 mgLVL-RecAP/kg/day, 8 mg LVL-RecAP/kg/day, or 16 mg LVL-RecAP/kg/day.

FIG. 4: Serum creatinine concentrations in piglets suffering from renalischemic/reperfusion damage. Sham animals underwent the surgicalprocedure during which the left kidney was removed. However, renalocclusion and reperfusion were not conducted. In the group that received0.32 mg/kg/day, on day 0, half of the dose was administered prior toreperfusion and the remaining half of the dose was administered at 8±2hours post-reperfusion. A full dose was administered daily for theremainder of the in-life period.

FIG. 5: AP concentrations in piglets suffering from renalischemic/reperfusion damage. Sham animals underwent the surgicalprocedure during which the left kidney was removed. However, renalocclusion and reperfusion were not conducted. In the group that received0.32 mg/kg/day (200 U/kg/day), on day 0, half of the dose wasadministered prior to reperfusion and the remaining half of the dose wasadministered at 8±2 hours post-reperfusion. A full dose was administereddaily for the remainder of the in-life period.

FIG. 6A-6D: Improvement of survival and body weight of Alp1^(−/−) miceby LVL-RecAP treatment. 6A) LVL-RecAP16 survived to the end of theexperiment. Average of survival was p19, p22 and p42.5 for Vehicle,LVL-RecAP1, and LVL-RecAP8, respectively. 6B) Alp1^(−/−) vehicle treatedmice are lighter than WT littermates; LVL-RecAP1 treatment improves bodyweight close to WT at p18. 6C) Vehicle treated mice do not survivelonger and LVL-RecAP8 treated mice are still lighter than WT littermatesat p53. 6D) Whereas LVL-RecAP16 treated mice present a body weight notsignificantly different from their WT littermates at p53.

FIG. 7A-7B: 7A) LVL-RecAP treatment improves the skeletal phenotype ofAlp1^(−/−) mice. Radiographs of spine, forelimb, rib cage, hindlimb andpaws from Akp2^(−/−) mice treated with vehicle, LVL-RecAP1, LVL-RecAP8,LVL-RecAP16, and untreated WT controls (magnification 5×). A) Akp2^(−/−)mice display a severe osteomalacia (arrows) in vertebrae, long bones andrib cage. Secondary ossification centers are missing in Akp2^(−/−) mice(asterisk). Treatment with LVL-RecAP1 slightly corrects that phenotype(arrowheads) compared to untreated WT at p18. 7B) Treatment withLVL-RecAP8 and LVL-RecAP16 clearly corrects the bone phenotype invertebrae, long bones and rib cage. Specifically the distal mostextremities are improved as the development of the secondaryossification centers in the metatarsal region demonstrates (arrowheads).

FIG. 8A-8C: 8A) Improved mineralization in LVL-RecAP-treated Alp1^(−/−)mice. A) Histological analysis of femora of vehicle treated Alp1^(−/−)(p18), LVL-RecAP8 (p51), LVL-RecAP16 p53, and untreated WT mice p53. VonKossa staining revealed better bone mineralization with increaseddosages LVL-RecAP8 and LVL-RecAP16. Secondary ossification centers andcortical bone region show a striking improvement in mineralization(black) compared with untreated. There is less trabecular bone inLVL-RecAP8 and LVL-RecAP16 compared to WT, but increased mineralizationin the trabecular region as well as more osteoid, expected to becometrabecular bone. 8B/8C) Analysis of BV/TV and OV/BV for LVL-RecAP8 andLVL-RecAP16 demonstrated less bone volume and higher osteoid volume thanage-matched controls.

FIG. 9A-9C: Improvement of osteomalacia and plasma PP_(i) levels inLVL-RecAP-treated Alp1^(−/−) mice. 9A) Histological analysis of femoraof vehicle treated Alp1^(−/−) (p18), LVL-RecAP8 (p51), LVL-RecAP16 p53,and untreated WT mice p53. Goldner's Trichrome staining of the femorasections show severe osteomalacia in Alp1^(−/−), and improvedmineralization in the cortical area and in the secondary ossificationwith increased dosages LVL-RecAP8 and LVL-RecAP16. Presence of enlargedareas of osteoid, suggests deposition of bone, but not yet mineralized.9B/9C) PP_(i) concentrations in the plasma of Akp2^(−/−) mice receivingLVL-RecAP1, LVL-RecAP16, and WT. LVL-RecAP1 treatment leads to asignificant reduction of the elevated PP_(i) levels in Alp1^(−/−) miceat p18. At p53 LVL-RecAP16 treatment resulted in a correction of plasmaPP_(i) levels comparable to WT controls.

FIG. 10A-10F: Absence of craniofacial defects in LVL-RecAP-treatedAlp1^(−/−) mice. μCT isosurface images of WT (10A, 10D), vehicle-treatedAlp1^(−/−) (10B, 10E) and LVL-RecAP16 (10C, 10F) mouse skulls at p21 andp53, respectively. Neither frontal nor parietal bones of treatedAlp1^(−/−) mice were significantly different from those of WT mice.Adult skulls of LVL-RecAP16-treated Alp1^(−/−) mice did not appeardifferent from WT in terms of size and shape.

FIG. 11A-11Q: LVL-RecAP treatment partially rescues the dentoalveolarphenotype in Alp1^(−/−) mice. Compared to (11A) radiography and (11D)μCT analysis of wild-type controls at p25-26, (11B, 11F) untreated mousemandibles feature hypomineralized and reduced alveolar bone (11A/11B),short molars (M1, M2, and M3) with thin dentin (11D/11E) and wide pulpchambers, and defective (white asterisk) incisor (INC) dentin. Comparedto (11E) histology of control periodontal tissues, (11G) Alp1^(−/−) miceexhibit no acellular cementum (11A/11C), and alveolar bone osteoidinvades the PDL space, creating bone-tooth ankylosis (asterisk). (11C,11H) LVL-RecAP8-treated Alp1^(−/−) mice show improved radiographicappearance of molar height, dentin thickness, and bone mineralization,though the incisor teeth remain defective. (11I) HistologicallyLVL-RecAP does not restore acellular cementum to the root surface.Compared to (11J, 11L) controls at p53, (11K, 11M) LVL-RecAP16-treatedAlp1^(−/−) mice exhibit reduced alveolar bone mineralization aroundmolar teeth. Molar form and mineralization appear relatively normal intreated Alp1^(−/−) mice, while incisor teeth remain severely affected onthe root analogue (white asterisk). Compared to histology of (11N) WTcontrol molars, (11P) LVL-RecAP16-treated Alp1^(−/−) mice feature a mixof mineralized alveolar bone and osteoid (asterisks), and a reduced butmaintained PDL space. Poor periodontal attachment is evidenced by lackof cementum, PDL disorganization and detachment, and down growth of thejunctional epithelium. Small regions of PDL attachment (chevron) to thetooth were noted. Compared to the strong and parallel organization ofPDL collagen fibers in (11O) control tissues, indicated by picrosiriusstaining under polarized light, (11Q) LVL-RecAP16-treated Alp1^(−/−)mice feature a less organized PDL, though regions of organization andattachment exist adjacent to breakthrough areas of tooth root attachment(white chevron).

FIG. 12A-12L: RecAP treatment attenuates the LPS-induced cytokineproduction in human proximal tubule epithelial cells (ciPTEC). CiPTECwere pre-treated with recAP (1-5-10 U/ml) followed by LPS-incubation (10μg/ml) for 24 hrs and subsequently TNF-α, IL-6 and IL-8 production wasmeasured (12A, 12B, 12C) on gene level in cells by qPCR (10 U/ml recAP)and (12D, 12E, 12F) on protein level in supernatant by ELISA. (12G, 12H,12I) recAP (10 U/ml) was administered 2 hrs preceding LPS exposure,simultaneously with LPS or 2 hrs after LPS exposure, followed bymeasurement of TNF-α, IL-6 and IL-8 protein content. (12J, 12K, 12L)ciPTEC were pre-treated with inactive AP for 2 hrs, lacking hydrolyzingproperties, followed by LPS incubation (10 μg/ml) for 24 hrs, whereafterTNF-α, IL-6 and IL-8 protein content was measured. Control cells wereincubated with culture media. Data is expressed as mean±SEM (n=5),#p<0.05 compared to control, * p<0.05 compared to LPS.

FIG. 13A-13F: The effects of recAP are not restricted to LPS-inducedinflammation and are renal specific. CiPTEC were pre-treated with recAP(10 U/ml) for 2 hrs followed by a 24-hour incubation with (13A, 13B)TNF-α (10 ng/ml) or (13C, 13D) supernatant of LPS-stimulated peripheralblood mononuclear cells (PBMCs, 1 ng/ml LPS), whereafter IL-6 and IL-8production was measured on protein level by ELISA. (13E, 13F) PBMCs werepreincubated for 2 hrs with recAP (10 U/ml) followed by LPS exposure (1ng/ml) for 24 hrs. IL-6 and TNF-α production was measured by ELISA.Control cells were incubated with culture media. Data is expressed asmean±SEM (n=5), #p<0.05 compared to control, * p<0.05 compared to LPS.

FIG. 14: The effect of recAP on LPS-induced ATP release in vitro. ciPTECwere pre-treated with recAP (10 U/ml) followed by LPS-incubation (10μg/ml or 100 μg/ml). After 30 minutes, supernatant was collected todetermine cellular ATP release by bioluminescence. Control cells wereincubated with culture media. Data is expressed as mean±SEM (n=5),#p<0.05 compared to control.

FIG. 15A-15D: RecAP prevents the LPS-induced deterioration of renalfunction in vivo. Renal function was assessed by the transcutaneousmeasurement of FITC-sinistrin t_(1/2). AKI was induced in rats by LPS(0.3 mg/kg b.w., t=0), followed by recAP treatment (1000 U/kg b.w.) att=2. T_(1/2) measurements were performed at t=2 hrs and t=21.5 hrs (15A,15B: examples of FITC-sinistrin kinetics obtained for one rat at the tworespective time-points). Urine was collected between t=5 and t=16 andplasma was sampled at t=1.5 and t=24 hrs, allowing calculation of (15C)Fractional Urea excretion and (15D) Creatinine Clearance with theaverage plasma value of t=1.5 and t=24. Data is expressed as mean±SEM(Placebo, LPS n=6; LPS+recAP n=5), #p<0.05 compared to placebo.

FIG. 16A-16E: RecAP prevents renal injury during LPS-induced AKI invivo. (16A) Urinary KIM-1 excretion and (16B) NGAL excretion, (16C)plasma NGAL levels (t=24), and (16D) renal KIM-1 protein content weredetermined by ELISA. (16E) Paraffin kidney sections were stained withanti-KIM-1 to visualize KIM-1 protein expression. Scale bars: 400 μm(left panel), 200 μm (right panel). Data is expressed as mean±SEM(Placebo, LPS n=6; LPS+recAP n=5; urinary parameters: Placebo n=5),#p<0.05 compared to placebo.

FIG. 17A-17C. No effect of recAP on LPS-induced cytokines insupernatant. Supernatant of 24 hrs LPS-incubated (10 μg/ml) ormedium-incubated (Control) ciPTEC was collected, incubated with orwithout recAP (10 U/ml) for another 24 hrs followed by the measurementof TNF-α, IL-6 and IL-8 (17A, 17B, 17C) by ELISA. Data is expressed asmean±SEM, #p<0.05 compared to control, * p<0.05 compared to LPS.

FIG. 18. Correlation between enzyme activities of RecAP in human serumat 25° C. and at 37° C.

FIG. 19. Correlation between enzyme activities of LVL-RecAP in humanserum at 25° C. and at 37° C.

FIG. 20 shows the activity/μg protein at 25° C. and at 37° C. for RecAPand LVL-RecAP. Values represent mean Δactivity between 25° C. and 37° C.for a given protein concentration.

EXAMPLES Example 1 Stability of LVL-RecAP in Buffer Materials:

Human recombinant alkaline phosphatases:

-   -   1. sALPI-ALPP-CD (SEQ ID NO: 4) (DOM: 4 Aug. 2008)    -   2. LVL-recAP (SEQ ID NO: 1) (DOM: 14 Oct. 2011)

Methods: Zinc Dependency:

The enzyme activity and protein content of both human recombinantalkaline phosphatase batches were determined. Subsequently 100 μg/mLprotein solutions were prepared for each condition to determine Zincdependency for batches sALPI-ALPP-CD and LVL-recAP as outlined in Table2.

The prepared samples were stored at RT and analysed for enzyme activityat T=0, T=2 h and T=24 h.

TABLE 2 Conditions of recombinant alkaline phosphatase batchessALPI-ALPP-CD and LVL-recAP to determine their stability (retainedactivity) in the presence and absence of Zinc and the influence of achelator (EDTA). Zn BSA Mg Mannitol EDTA Condition (μM) (%) (mM) (%)(mM) 1 0 0 1 1 0 2 0.01 0 1 1 0 3 10 0 1 1 0 4 100 0 1 1 0 5 1000 0 1 10 6 0 0 1 1 2 7 0 0 1 1 5 8 0 0 1 1 10 9 0 0 1 1 100 10 50 0.025 1 1 0

Determinations of enzyme activities were according to standardprocedures, as indicated in SOP PC001.

Protein concentrations were determined by OD₂₈₀ measurements (ε_(recAP)1.01 mL/mg/cm OD₂₈₀)

Results

TABLE 3 activity of the different alkaline phosphatases under theconditions as specified in Table 2. sALPI-ALPP-CD LVL-RecAP Diluent t =0 t = 2 t = 24 t = 0 t = 2 t = 24 1 45.7 53.3 50 60.7 59.8 57.4 2 48.253.8 51.6 57.7 57.2 58.1 3 47.3 54.4 51.6 57.7 54.2 52.9 4 46.1 52 53.455.3 53.7 55.7 5 48.2 51.2 52.2 56.7 56.3 54.7 6 46.9 24.2 18.7 57.654.2* 37.7* 7 47 23.6 20.4 57.6 52.1* 38.9* 8 45 23.2 17.2 56.8 50.3*35.8* 9 50.9 22.5 16.2 55.6 46.5* 31.2* 10 47.3 55.1 53.6 62.7 57.5 55.4

As clearly shown in Table 3, LVL-RecAP is considerably more stable (i.e.shows more residual enzyme activity; denoted by values with asterisks)in the presence of a metal chelating agent (EDTA) than sALPI-ALPP CD,implicating less dependency of Zn²⁺ for its activity.

Example 2

Stability of LVL-RecAP in buffer

Materials:

Human recombinant alkaline phosphatases:

-   -   1. sALPI-ALPP-CD (DOM: 4 Aug. 2008)    -   2. LVL-recAP (DOM: 14-Oct. 2011)

Methods

In a second independent experiment, stability of sALPI-ALPP-CD andLVL-recAP were tested for several conditions as outlined in table 4.

For both recombinant AP batches in this experiment 100 μg/mL proteinsolutions in 0.025M glycine buffer pH 9.6, human serum and human citrateplasma were prepared for each buffer condition to determine stability.

All the prepared recombinant AP samples were incubated at 37° C. andanalysed for enzyme activity at T=0, T=0.5 h, T=1 h, T=2 h and T=24 h.

TABLE 4 Conditions of recombinant alkaline phosphatase batches sALPI-ALPP-CD and LVL-recAP in glycine buffer to determine their stability(retained activity) in the presence/absence of Zinc and the influence ofa Chelator (EDTA or Citrate). Zn BSA Mg Mannitol EDTA Condition (μM) %(mM) (%) (mM) 1 0 0 1 1 0 2 0 0 1 1 2 3 0 0 1 1 10 4 0 0 1 1 100 8 500.025 1 1 0

Results

TABLE 5 activity of the different alkaline phosphatases under theconditions as specified in Table 4. LVL-recAP sALPI-ALPP-CD Condition t= 0 t = 0.5 t = 1 t = 2 t = 24 t = 0 t = 0.5 t = 1 t = 2 t = 240 1 55.157.7 54.9 55.2 55.8 63 52.5 53.8 53.8 55.2 2 59.5 52.5 52 51.6* 37.7*56.5 48 51.6 36.3 23.3 3 50.9 53.5 51.6 49.2* 35.2* 3.3 44.7 43.7 32.918.9 4 49.3 43.9 48.7 41.3* 24.2* 49.8 37.9 34.6 29.4 13.3 8 12.2 49.353.9 53.1 52.1 55.9 56 57.9 57.5 56

Determinations of enzyme activities were performed according to SOPPC001. Protein concentrations were determined by OD280 measurements(εrecAP 1.01 mL/mg/cm OD280).

As clearly shown in Table 5 and in line with results in Table 3,LVL-RecAP is considerably more stable in the presence of a metalchelating agent (EDTA) than sALPI-ALPP CD (as denoted by values withasterisks), implicating less dependency of Zn²⁺ for its activity.

Example 3

Heat stability and PLP kinetics of LVL-RecAP

Methods Protein Expression

Expression plasmids containing secreted, FLAG epitope-taggedsALPI-PLAP-CD and LVL-RecAP were constructed as described previously(Kozlenkov et al. J Biol Chem 277, 22992-22999 (2002) and Kozlenkov etal. J. Bone Miner. Res. 19, 1862-1872 (2004)). The FLAG-tagged enzymeswere transiently transfected into COS-1 cells by electroporation, thengrown in DMEM medium for 24 h, as previously described (Narisawa et al.Am. J Physiol. Gastrointest. Liver Physiol. 293, G1068-1077 (2007)) whenthe medium was replaced with serum-free Opti-MEM (Life Technologies).Opti-MEM containing secreted proteins was collected 60 hours aftertransfection, then filtered through a 2-μm cellulose acetate filter anddialyzed against TBS containing 1 mM MgCl₂ and 20 μM ZnCl₂.

Enzyme Kinetics

To measure the relative catalytic activities of FLAG-tagged enzymes,micro-titer plates were coated with anti-FLAG M2 antibody(Sigma-Aldrich) at 0.2-0.6 μg mL⁻¹. These plates were incubated withsaturating concentrations of FLAG-tagged LVL-RecAP or sALPI-PLAP for 3 hat room temperature, after which plates were washed with PBS, containing0.008% Tween-80 and the relative activities for PLP were compared forthe M2-saturated enzymes.

Hydrolysis of the physiological substrate pyridoxal-5′-phosphate (PLP)(Sigma-Aldrich) was measured at pH 7.4 in standard assay buffer (50 mMTris-HCl buffer, 100 mM NaCl, 1 mM MgCl₂ and 20 μM ZnCl₂). Theconcentration of released phosphate was determined using P_(i) ColorLockGold (Innova Biosciences) by measuring absorbance at 650 nm (A₆₅₀).Standard curves constructed for increasing concentrations of phosphatewere linear between 0-50 μM and all experiments were designed to fallwithin this range of hydrolyzed phosphate concentrations. Molar reactionrates, expressed as [P_(i)] s⁻¹ were calculated for the indicatedsubstrate concentration range and were fitted to a one-binding sitemodel (GraphPad Prism) versus [substrate] to calculate K_(m)(Lineweaver-Burk plots were not applied, because of lack of precision ofreciprocal conversions at very low substrate concentrations).

A substrate concentration for PLP of 400 μM was used. The soluble enzymeconcentration was about 1 nM and incubation times ranged from 15-30 min,depending on the catalytic efficiency for each enzyme. To ensuresteady-state conditions and to correct for non-specific substratesignals in the P_(i) ColorLock Gold method, an early reading (at 5 min)was subtracted from the later reading, and ΔA₆₅₀ was measured on thecorresponding P_(i) standard curve, constructed for each experimentseparately. All experiments were carried out three to five times and thederived constants are reported as mean±SD.

To measure the heat stability, FLAG-tagged enzymes were incubated at 65°C. in 1 M DEA (pH 9.8) containing 1 mM MgCl₂ and 20 μM ZnCl₂. Sampleswere removed at different time points and placed on ice, then residualactivity was measured using pNPP using the following method: Theactivity of bound enzymes was measured as the absorbance at 405 nm(A₄₀₅) as a function of time at 25° C., using pNPP (10 mM) as asubstrate, at pH 7.4 in 50 mM Tris-HCl buffer, 100 mM NaCl, containing 1mM MgCl₂ and 20 μM ZnCl₂. Enzymes were also incubated in this buffer for10 min at increasing temperatures (25-100° C.) and residual activity wasmeasured in the same manner.

Results Production of FLAG-Tagged Enzymes

To compare the kinetic properties of LVL-RecAP with sALPI-PLAP, aFLAG-tag sequence was added to both cDNAs, as done previously tocomparatively study PLAP and TNAP (Kozlenkov et al. J Biol Chem 277,22992-22999 (2002) and Kozlenkov et al. J. Bone Miner. Res. 19,1862-1872 (2004)). The cDNAs were expressed in COS-1 cells and culturesupernatant containing secreted enzymes was recovered. Successfulexpression and recovery were confirmed by anti-FLAG antibody westernblot analysis.

Kinetics Parameters with Physiological Substrates

The phosphohydrolase properties of LVL-RecAP and sALPI-PLAP wereinvestigated for physiological substrates implicated in inflammation andseizures, specifically the vitamin B₆ vitamer PLP. LVL-RecAP showedlower Km than sALPI-PLAP (30.1 μM vs 60.7 μM) at physiologic pH (7.4),indicating that the improved LVL-RecAP has higher affinity for PLP thansALPI-PLAP at physiologic pH.

Enzyme Stability

The impact of the amino acid mutations in LVL-RecAP on the overallstability of the enzyme was investigated by heat inactivation studies.Although sALPI-PLAP has already highly improved heat resistance (50%inactivation at 77.8° C.), LVL-RecAP was even more resistant to heatinactivation (50% inactivation at 80.6° C.).

Example 4

Pharmacokinetic distribution of LVL-RecAP in rats

Part A Radiolabelling with Iodine-125

The sALPI-PLAP-CD protein and the LVL-RecAP protein were radiolabelledwith Iodine-125 using the chloramine-T procedure as described byGreenwood et al.* The principle of labelling is based on the “in situ”oxidation of iodide to atomic iodine and its nucleophilic substitutioninto phenol rings in ortho position of the hydroxyl group of tyrosineresidues.

The sALPI-PLAP protein was radioiodinated using the Chloramine-Ttechnique in order to obtain ˜0.5 mCi/mg final specific activity and ˜1mg/mL (NaCl 0.9%) final concentration. *F. C Greenwood, V. M Hunter, H.G Glover, The preparation of 131I labelled growth hormone of highspecific radioactivity, Biochem. J. 89 (1963) 114-123

A.1 Materials

-   -   sALPI-PLAP Alkaline Phosphatase (496.7 U/mg) and LVL-RecAP (624        U/mg) was provided by AM-Pharma at 6.34 mg/mL concentration in        25% glycerol w/v, 5 mM Tris, 2 mM MgCl₂, 50 μM ZnCl₂, pH 8.0.        sALPI-PLAP protein and LVL-RecAP protein were stored at 4° C.    -   Iodine-125 radionuclide was purchased from Perkin Elmer as        sodium iodide in 10⁻⁵N sodium hydroxide (specific activity:        643.8 GBq/mg—radionuclide purity: 99.95%).    -   Chloramine-T (N-chloro-p-toluenesulfonamide, MW 227.6 g/mol),        trichloroacetic acid, sodium metabisulfite (MW 190.1 g/mol) and        tyrosine were purchased from Sigma.

Tris Buffer 5 mM pH 8 was prepared in Chelatec laboratory.

-   -   NaCl 0.9% was provided by Versol®.

A.2 Method

850 μg of protein, about 600 μCi of Na¹²⁵I, 50 μL of Tris buffer and 10μL of chloramine-T (404.8 nmol, 50 equivalents/protein) weresuccessively added in a 1.5 mL Lo-Bind eppendorf tube. The reaction wasallowed to stir 1 minute at room temperature. 2 μL of the radiolabellingmedium was mixed with MBS 5% solution and the radiolabelling efficiencywas evaluated by Instant thin layer chromatography (ITLC) with 10% TCAas eluent (alkaline phosphatase at the bottom of the strip and free125-iodine is eluted at the top of the strip).

After adding 30 μL of tyrosine solution (10 mg/mL in water) to the crudemixture, the iodinated sALPI-PLAP and iodinated LVL-RecAP were purifiedby gel filtration (G10, GE Healthcare) eluted with NaCl 0.9%. Fractionsof 0.2 mL volume were collected in test tubes. Radioactivity in eachfraction was measured in an automatic Gamma Counter calibrated foriodine-125 radionuclide (Wallace Wizard 2470—Perkin Elmer). Thefractions containing the desired radioiodinated product were pooled. Theradiochemical purity of the radiolabelled compound was verified by ITLC.

A.3 Results and Characteristic of Radiolabelled sALPI-PLAP Protein

The efficiency of radiolabelling determined by ITLC was over 85% forboth proteins. After G10 purification, radiopurity of the labellingreaction is higher than 97% for both proteins.

The characteristics of radiolabelled sALPI-PLAP solution after G10purification are summarized in Table 6.

TABLE 6 Characteristics of ¹²⁵I-sALPI-PLAP solution after purification¹²⁵I-sALPI-PLAP ¹²⁵I-LVL-RecAP Labelling efficiency 86.36% 85.64% (ITLC)G10 purification Concentration 1.149 1.145 (mg/mL) Specific Activity0.669 0.544 (mCi/mg) Volumic Activity 0.791 0.637 (mCi/mL) Radiopurity(%) 97.23 97.8

A.4 Alkaline Phosphatase Activity

The enzymatic activity of radiolabelled sALPI-PLAP was assessed viaELISA.

Materials

-   -   Alkaline phosphatase colorimetric assay kit (reference        ab83369—batch number: GR118166-3).    -   Unlabelled recombinant human alkaline phosphatase diluted at        0.25 mg/mL in NaCl 0.9%    -   Radiolabelled recombinant human alkaline phosphatase diluted at        0.25 mg/mL in NaCl 0.9%

Protocol

The Abcam kit uses p-nitrophenyl phosphate (pNPP) as a phosphatasesubstrate which turns yellow (Lamda max=405 nm) when dephosphorylated byAP. The kit can detect 10-250₁113 AP in samples.

The alkaline phosphatase colorimetric assay was performed on unlabelledand radioiodinated sALPI-PLAP. The assay was performed as described inprotocol provided by Abcam.

Briefly, a standard curve was generated from 0 to 20 nmol/well of pNPPstandard (final volume: 120 μL). 10 μL of AP enzyme solution was addedin each well. In parallel, the test sample of sALPI-PLAP was diluted15000 and 8000 times in assay buffer. 10 and 20 μL of each dilution wasadded and the final volume was brought to 80 μL with assay buffer. Then,50 μL of pNPP solution was added to each well containing the testsample.

The standard and sample reactions were incubated 60 min at 25° C. in thedark. All reactions were then stopped with 20 μL of stop solution. TheO.D. at 405 nm was measured in a microplate reader.

pNP standard curve was plotted. The sample readings were applied to thestandard curve to get the amount of pNP generated by AP sample. APactivity of the test samples can be calculated:

sALPI-PLAP activity (U/mL)=A/V/T×dilution factor of test sample

A: amount of pNP generated by samples (in μmol)V: volume of sample added in the assay well (in mL)T: reaction time in minutes

sALPI-PLAP activity (U/mg)=sALPI-PLAP activity (U/mL)/concentration ofsALPI-PLAP (mg/mL)

Results

Table 7 summarises the results.

TABLE 7 Enzymatic activity of unlabelled sALPI-PLAP and ¹²⁵I-ALPI-PLAPsALPI-PLAP activity (U/mg) Dilution 1/15000 Dilution 1/8000 MeansALPI-PLAP 474.6 471.6 473.1 ¹²⁵I-sALPI-PLAP 482.6 475.3 479.0 LVL-RecAP501.3 530.2 515.8 ¹²⁵I-LVL-RecAP 539.2 588.5 563.8

The enzymatic activity of unlabelled and radiolabelled sALPI-PLAP issimilar to about 475 U/mg. Thus, the activity of sALPI-PLAP is notcompromised by the radioiodination using chloramine-T as oxidant.

Part B: Biodistribution Study of ¹²⁵I-sALPI-PLAP Protein in Healthy Rats

B.1 Materials

The characteristics of the rat strain used in this study are presentedbelow:

-   Species: Sprague Dawley Rats-   Strain: Crl CD® (SD) IGS BR-   Source: Charles RIver France-   Number and sex: 15 males-   Body weight range/age: Approximately 250 g at the beginning of the    study-   Acclimation period: Five days before treatment-   Identification method: Cage identification with the group (sacrifice    time).

Animal Management

-   Husbandry: The rats were housed in animal facilities before    treatment and in radioactivity room after treatment.-   Food: Freely available rat diet. No fasting period before treatment.-   Water: Tap water was delivered by polypropylene bottle ad libitum.-   Housing: Before treatment, animals were housed in groups of three in    polycarbonate cages in standard conditions, identified by a card    indicating the study number, the animal number, the sex, and the    dates of beginning and end of the study. Animals designated for    excretion balance were transferred in metabolism cages (one by cage)    after treatment.-   Environmental: The temperature was recorded every day. The    temperature of the room was between 22 and 24° C. The artificial    light cycle was controlled using an automatic timer (10 hours of    light, 14 hours of dark).-   Personnel: Associates involved were appropriately qualified and    trained.-   Selection: Animals were examined at receipt by the study director.    Only healthy animals were selected. Particular attention was paid to    any sign of inflammatory reaction in the animals (e.g. abscess; skin    inflammation etc).

B.2 Dosing Solution of ¹²⁵I-sALPI-PLAP and 125I-LVL-RecAP

The Iodinated sALPI-PLAP and LVL-RecAP solutions were diluted at 0.25mg/mL in NaCl 0.9% just prior to in vivo administration.

B. 3 Study Design

The study design is presented in Tables 8 and 9.

TABLE 8 Study design for ¹²⁵I-sALPI-PLAP in vivo distribution Number ofBlood sampling Sacrifice Group animals time time Biodistribution A1 3males 2 mn, 10 mn 30 mn Blood and organs A2 3 males 5 mn, 45 mn 2 hBlood and organs A3 3 males 15 mn, 4 h 6 h Blood and organs A4 3 males 1h, 3 h, 18 h 24 h Blood and organs  A5* 3 males NA 48 h Blood and organs*housed individually in metabolism cages with urine and faecescollection at 24 h and 48 h

TABLE 9 Study design for ¹²⁵I-LVL-RecAP in vivo distribution Number ofBlood sampling Sacrifice Group animals time time Biodistribution B1 3males 2 mn, 10 mn 30 mn Blood and organs B2 3 males 5 mn, 45 mn 2 hBlood and organs B3 3 males 15 mn, 4 h 6 h Blood and organs B4 3 males 1h, 3 h, 18 h 24 h Blood and organs  B5* 3 males NA 48 h Blood and organs

B.4 Administration

At the time of the experiment, the mean weight of the Sprague Dawleyrats was about 240 g. Unanesthetized rats were injected intravenously inlateral tail vein (left) at a dose level of 400 μg/kg corresponding to aquantity of 96 μg protein per rat and an activity of about 58 μCi perrat. The volume of injection was about 380 μL. The individual dosevolumes were calculated using individual body weight of each rat on theday of treatment. Rats were placed in a contention device. In order todilate the tail blood vessels, the tail was dipped in warm water (45°C.) and then disinfected with alcohol. The dosing solution was slowlyinjected in the tail vein. In order to calculate the actual dosereceived by each rat, the syringes were weighed before and aftertreatment and an aliquot of dosing solution was counted in a GammaCounter.

B.5 Distribution in Various Organs

At the time of sacrifice, the animals were anaesthetized byintraperitoneal injection of 2.5 mL/kg body weight of a mixture ketaminehydrochloride (50 mg/mL) and xylazine hydrochloride (20 mg/mL) in PBS.Rats were then rapidly sacrificed by exsanguination via intracardiacpuncture. The organs of interest were harvested followed by the cuttingin pieces for organs weighting more that 2 g such as liver, stomach,small intestine and colon. Each piece was thereafter rinsed withphysiological serum prior to be wiped with soft paper tissue, weighedand counted separately. The selected organs were liver, kidneys, heart,lungs, spleen, skeletal muscle, femur, brain, thyroid, stomach, smallintestine with content, colon with content, skin and perirenal fat.

The counting of tissue radioactivity was performed in an automatic gammacounter (Wallace Wizard 2470—Perkin Elmer) calibrated for Iodine-125radionuclide (efficiency: 74%—counting time: 10 sec).

The concentration of radioactivity in the organs/tissues is expressed aspercentage of the injected dose per gram of tissue (% ID/g). Dataanalysis includes the percentage of the injected dose (% ID) andequivalent quantity of protein per organ or tissue. For well-definedorgans, these were calculated using the radioactivity counted in wholeorgan. For blood, this was approached assuming that blood accounts for6.4% of total body weight.

In addition, the ratio between the radioactivity retained in the tissuesand the radioactivity in blood was calculated (ratio organ/blood).Finally, the ratio between the organ/blood ratio of sALPI-PLAP and theorgan/blood ratio of LVL-RecAP was calculated (FIG. 2).

B.6 Distribution in Rat Blood and Serum

Blood and Serum Radioactive Level

At the time of sacrifice, the blood samples were obtained fromexsanguinations via intracardiac puncture on anaesthetized rat byintraperitoneal injection of a mixture of ketamine hydrochloride andxylazine hydrochloride in PBS.

At the other time points indicated in the study design (table 8 and 9),the blood was withdrawn from the lateral tail vein (right) using a23-gauge butterfly needle without anesthesia.

Each blood sample was collected into preweighed Microvette tubes withclotting activator (Sarstedt). The tubes were weighed and theradioactivity was measured in the Gamma counter.

Blood samples were incubated at room temperature for 30 min and then,they are centrifuged for 5 minutes at 10 000 g to prepare serum. Serumwas collected into pre-weighed tubes and counted in the Gamma counter.

The concentration of radioactivity in blood and serum is expressed aspercentage of the injected dose and equivalent quantity of sALPI-PLAPper mL.

Data analysis includes the percentage of the injected calculated for thetotal blood and serum.

Tables 10 and 11 show the ratio of radioactivity measured in differentorgans in relation to blood serum radioactivity of sALPI-PLAP andLVL-RecAP respectively. FIG. 2 shows the ratio of the organ/blood ratioLVL-RecAP to organ/blood ratio sALPI-PLAP. A ratio of 2, for instance,indicates that LVL-RecAP is targeted twice as much to the indicatedorgan as sALPI-PLAP. Higher ratios further indicate that relatively moreactivity of LVL-RecAP is found in a particular organ when the same doseis used.

TABLE 10 Organ/Blood ratio for sALPI-PLAP ORGANS 0.5 H 2 H 6 H 24 H 48 HThyroid/ 0.1624 ± 0.0157 4.6771 ± 0.4433 31.745 ± 5.175 208.16 ± 23.74 733.85 ± 79.16  Trachea Skin 0.0307 ± 0.0035 0.2842 ± 0.0134 0.4661 ±0.1617 0.9448 ± 0.2565 1.0787 ± 0.3686 Kidneys 0.2076 ± 0.0011 0.3438 ±0.0310 0.3535 ± 0.0219 0.3250 ± 0.0467 0.4299 ± 0.0219 Stomach 0.0446 ±0.0016 1.0699 ± 0.1665 3.0622 ± 0.6977 1.4478 ± 0.1200 3.5819 ± 0.9018Spleen 0.5388 ± 0.0796 0.4133 ± 0.0322 0.2880 ± 0.0283 0.2256 ± 0.02700.2273 ± 0.0254 Liver 5.2403 ± 0.1943 1.9917 ± 0.0347 0.4811 ± 0.08770.3219 ± 0.0325 0.4676 ± 0.0295 Heart 0.1824 ± 0.0136 0.2259 ± 0.01980.2605 ± 0.0217 0.2791 ± 0.0387 0.2809 ± 0.0165 Lungs 0.1777 ± 0.01190.2767 ± 0.0318 0.3397 ± 0.0730 0.3833 ± 0.0091 0.3937 ± 0.0251 Skeletal0.0334 ± 0.0033 0.0978 ± 0.0193 0.0842 ± 0.0178 0.0876 ± 0.0038 0.0947 ±0.0150 muscle Fat 0.0095 ± 0.0023 0.0261 ± 0.0070 0.0509 ± 0.0111 0.0615± 0.0065 0.0874 ± 0.0153 Brain 0.0160 ± 0.0031 0.0257 ± 0.0011 0.0213 ±0.0011 0.0253 ± 0.0025 0.0408 ± 0.0063 Femur 0.1886 ± 0.0217 0.1991 ±0.0131 0.1901 ± 0.0155 0.1602 ± 0.0119 0.1612 ± 0.0132 Small 0.1882 ±0.0247 0.8440 ± 0.1896 1.1055 ± 0.0827 1.0729 ± 0.1268 0.6329 ± 0.1044intestine Colon 0.0227 ± 0.0028 0.0777 ± 0.0077 0.4150 ± 0.0489 0.3473 ±0.0596 0.3991 ± 0.0964

TABLE 11 Organ/Blood ratio for LVL-RecAP ORGANS 0.5 H 2 H 6 H 24 H 48 HThyroid/ 0.5934 ± 0.0892 7.3638 ± 1.5049 52.934 ± 13.475 462.83 ± 34.3951075.2 ± 118.65 Trachea Skin 0.0942 ± 0.0012 0.4110 ± 0.0485 0.6843 ±0.0810 1.2130 ± 0.1243 1.9213 ± 0.8965 Kidneys 0.5793 ± 0.0187 0.6425 ±0.0555 0.6428 ± 0.0098 0.8238 ± 0.0826 1.2481 ± 0.1089 Stomach 0.2262 ±0.1021 1.5983 ± 0.3099 6.2917 ± 3.5698 3.0042 ± 1.2012 0.9930 ± 0.3279Spleen 2.2771 ± 0.1172 1.0977 ± 0.1190 0.5831 ± 0.0292 0.3673 ± 0.07960.3623 ± 0.0429 Liver 7.6513 ± 0.9295 3.8409 ± 0.0277 0.6131 ± 0.09000.5869 ± 0.0967 0.7940 ± 0.0133 Heart 0.2030 ± 0.0105 0.2762 ± 0.00960.2780 ± 0.0316 0.2630 ± 0.0138 0.3144 ± 0.0023 Lungs 0.3007 ± 0.02010.3740 ± 0.0446 0.4332 ± 0.0111 0.3966 ± 0.0235 0.4515 ± 0.0220 Skeletal0.0462 ± 0.0072 0.0997 ± 0.0142 0.1036 ± 0.0251 0.0784 ± 0.0113 0.0953 ±0.0080 muscle Fat 0.0298 ± 0.0078 0.0380 ± 0.0063 0.0495 ± 0.0034 0.0781± 0.0155 0.0970 ± 0.0245 Brain 0.0261 ± 0.0052 0.0389 ± 0.0085 0.0253 ±0.0028 0.0288 ± 0.0016 0.0646 ± 0.0100 Femur 0.3764 ± 0.0102 0.2973 ±0.0098 0.2460 ± 0.0149 0.1804 ± 0.0325 0.1746 ± 0.0370 Small 0.1573 ±0.0049 0.5625 ± 0.0393 1.0308 ± 0.1134 0.9402 ± 0.0935 0.6612 ± 0.1003intestine Colon 0.0361 ± 0.0047 0.1049 ± 0.0271 0.3658 ± 0.0693 0.5353 ±0.1967 0.4787 ± 0.0712

Example 5

Akp2−/− Mouse Model of Infantile Hypophosphatasia

The Akp2−/− mice model of infantile hypophosphatasia is known in the art(J Dent Res 90(4):470-476, 2011). In short, Akp2−/− mice were created byinsertion of the Neo cassette into exon 6 of the mouse TNALP gene (Akp2)via homologous recombination to functionally inactivate the Akp2 gene,resulting in no detectable TNALP mRNA or protein.

Animal use and tissue collection procedures followed approved protocolsfrom the

Sanford-Burnham Medical Research Institute Animal Ethics Committee.Animals were treated with either vehicle (N=10), 1 mg LVL-RecAP/kg/day(N=10); 8 mg LVL-RecAP/kg/day (N=8) or 16 mg LVL-RecAP/kg/day (N=10).Survival was measured and skeletal development assessed. Mineralizationof kidneys was assessed. Plasma PPi, plasma pyridoxal, plasma calciumand phosphate levels were measured, femur and/or tibia length for thedifferent treatment groups were measured. MicroCT data was colleted foranalysis of residual hyperosteodosis at each dose.

Results

-   -   Enhanced long-term survival for animals treated at 16 mg/kg/day        (FIG. 3).    -   There was (incomplete) rescue of the skeletal defect in the long        bones even at the highest dose (data not shown)    -   There was some rescue in the dental phenotype (data not shown).    -   There seems to be rescue of craniosynostosis (data not shown; to        be confirmed)    -   The work on the kidneys is still ongoing. There was indication        of mineral in kidneys of untreated mice and less mineral in        kidneys of treated mice (data not shown).

Example 6

Ischemia Reperfusion in a pig kidney model

Materials and Methods Vehicle, Control and Test Article InformationControl and Test Article Preparation

Fresh control article, LVL-RecAP Diluent Solution (placebo), wasprepared for use on study prior to each dose administration and wasstored refrigerated at 2 to 8° C. when not in use.

The test article, LVL-RecAP, was used as received. No adjustment wasmade for purity when preparing the test article formulations.Formulations of the test article were prepared by mixing with theappropriate volume of sterile saline to achieve nominal concentrationsof 0, 0.96, or 4.8 mg/mL. Formulations were prepared prior to each doseadministration under a laminar flow hood using sterile equipment andaseptic techniques. The formulations were dispensed into the appropriatenumber of amber glass serum bottles, kept on ice prior to use and wereused for dosing within 2 hours of preparation. On occasion, additionalpreparations were made as necessary during the course of the study.

Analysis of Dosing Formulations

Duplicate 0.5 mL samples of the final dosing formulation were collectedon prior to dosing each day of preparation on Day 0 (Groups 3 to 7) andDay 7 (Group 7). Samples were collected from the middle strata and werestored frozen (−50 to −90° C.) for possible future analysis.

Test System Information Animal Acquisition and Acclimation

Male experimentally naïve Domestic Yorkshire crossbred swine (farm pigs)(approximately 8 to 10 weeks of age, at receipt) were received fromMidwest Research Swine, Gibbon, Minn. During the 10 to 28 dayacclimation period, the animals were observed daily with respect togeneral health and any signs of disease. Ova and parasite evaluations onstool samples were performed, and all results were negative for animalsplaced on study.

Randomization, Assignment to Study, and Maintenance

Using separate simple randomization procedures animals (weighing 12.5 to25.0 kg at randomization) were assigned to the control and treatmentgroups identified in the following Table 12.

TABLE 12 Group Assignments Group Number of Male Animals^(a) Number DoseLevel Initial Evaluated 1 Control 7 6 2 Sham^(b) 7 6 5 0.32 mg/kg 7 6 6 1.6 mg/kg 7 7 7 0.32 mg/kg/day^(c) 7 7 ^(a)On Day 0, animals underwenta surgical procedure during which the left or right kidney was removedand the contralateral renal artery was occluded for 45 minutes.Following occlusion, the vessel was allowed to reperfuse. Seven animalswere submitted for surgery in each group with the intent to achieve sixanimals on study. ^(b)Sham animals underwent the surgical procedureduring which the left kidney was removed. However, renal occlusion andreperfusion were not conducted. ^(c)On Day 0, half of the dose wasadministered prior to reperfusion and the remaining half of the dose wasadministered at 8 ± 2 hours post-reperfusion. A full dose wasadministered daily for the remainder of the in-life period.

Animals selected for study were as uniform in age and weight aspossible. A veterinarian assessed the health of the animals prior toplacement on study. Extra animals obtained for the study, but not placedon study, were transferred to the stock colony.

Each animal was assigned an animal number used in the Provantis™ datacollection system and was implanted with a microchip bearing a uniqueidentification number. Each animal was also identified by a vendor eartag. The individual animal number, implant number, ear tag number, andstudy number comprised a unique identification for each animal. Eachcage was identified by the animal number, study number, group number,and sex.

The animals were individually housed in runs with raised flooring orstainless steel mobile cages with plastic coated flooring. This type ofhousing provided adequate room for exercise for these animals. Animalenrichment was provided according to MPI Research SOP. Fluorescentlighting was provided for approximately 12 hours per day. The dark cyclewas interrupted intermittently due to study related activities.Temperature and humidity were continuously monitored, recorded, andmaintained to the maximum extent possible within the protocol designatedranges of 61 to 81° F. and 30 to 70%, respectively. The actualtemperature and humidity findings are not reported, but are maintainedin the study file.

Diet (Certified Lab Diet® #5K99, PMI Nutrition International, Inc.) wasoffered via limited feedings, except during designated periods. Foodenrichment, including fiber bits or tablets, was offered as needed.

Surgical Procedures Procedure-related Medications

The following Table 13 presents the procedure-related medications anddose levels used during the course of the study.

TABLE 13 Procedure-related Medications and Dose Levels Interval, DoseLevel, and Route Surgery Daily Medication (Day 0) PostsurgeryAcepromazine maleate 0.1 mg/kg IM — Atropine sulfate 0.05 mg/kg IM —Telazol 5 to 8 mg/kg IM — Isoflurane To effect by — inhalationBuprenorphine 0.02 mg/kg IM TID 0.02 mg/kg IM TID x 3 days Ketoprofen 3mg/kg IM 3 mg/kg IM SID x 3 days Cefazolin 25 mg/kg IV — Ceftiofur 2.2mg/kg IM 2.2 mg/kg IM SID x 3 days Lactated Ringer's 10 to 15 mL/kg/hr —solution (LRS) IV Marcaine 2 mg/kg INF — 0.9% NaCl As needed for —irrigation IV—Intravenous IM—Intramuscular INF—Infused into incisionSID—Once daily TID—Twice Daily (every 6 to 9 hours)

Pre- and post-operative procedures were conducted in accordance with MPIResearch SOP. The animals were fasted for at least 8 hours prior tosurgery and anesthesia was induced and maintained as indicated in Table13. Body temperature was maintained at 37±3° C. Prior to surgery,ultrasounds were performed to determine if renal cysts were present. Ifno cysts were observed, the left kidney was removed and the right kidneywas treated as described below. If cysts were present on one kidney,that kidney was removed and the contralateral kidney underwent theocclusion procedure. If cysts were present in both kidneys, the animalwas removed from study without undergoing the surgical procedure.

Surgical Procedure

Renal ischemia/reperfusion injury was induced using the procedurepublished by Lee et al (J. Vet. Med. Sci 72(1): 127-130). Onceanesthetized, all animals were placed in dorsal recumbency and thesurgical sites were prepared with alternating wipes of chlorhexidinescrub and solution. A midline laparotomy was performed to expose bothkidneys. Based on the ultrasound findings, the left or right kidney wasremoved.

For Group 2 animals (Sham), the inserted lap sponges were then removedand accounted for and the abdomen was lavaged with warm sodium chloride.The abdomen was closed in a routine manner and the skin was closed withskin staples and tissue glue. The animals were then allowed to recover.

For all other animals, after removal of the designated kidney, theremaining renal vessels were isolated and retracted using vessel loops.The vessel loops were used to occlude the vessels for 45 (±1) minutes,after which the vessels were allowed to reperfuse. An intravenous bolusdose of the control or test article was administered at a dose volume of0.333 mL/kg (a half dose of 0.167 mL/kg was administered to Group 7animals) just prior to reperfusion. For all Group 1, 5 and 6 animals(control, 0.32, and 1.6 mg/kg), the implanted lap sponges were thenremoved and accounted for, the abdomen was lavaged and closed asdescribed above, and the animals were allowed to recover. For all Group7 animals (0.32 mg/kg/day), an incision was made in the groin and theleft or right femoral vein was isolated. A catheter was advanced intothe vessel and the catheter was tunneled under the skin and exteriorizedthrough an incision made on the thorax. A port was attached and anchoredto the muscle with non-absorbable suture. The implanted lap sponges werethen removed and accounted for, the abdomen was lavaged and closed asdescribed above, and the animals were allowed to recover.

Test or Control Article Administration

On Day 0, the control or test article was intravenously administered toall Group 1, 5, and 6 animals just prior to reperfusion at a full doseof 0, 0.32, and 1.6 mg/kg, respectively. All doses were administered ata dose volume of 0.333 mL/kg. Also on Day 0, the test article wasintravenously administered to all Group 7 animals at a combined doselevel of 0.32 mg/kg in two separate half doses of 0.16 mg/kg at a dosevolume of 0.167 mL/kg. The first dose was administered just prior toreperfusion and the second dose was administered at approximately 8 (±2)hours post-reperfusion. Full doses of 0.32 mg/kg/day (0.333 mL/kg) wereadministered to all Group 7 animals on Days 1 to 7 at approximately thesame time of the day as the initial Day 0 dose (±2 hours).

Statistics

Table 14 below defines the set of comparisons used in the statisticalanalyses described in this section.

TABLE 14 Table of Statistical Comparisons Reference Comparison GroupGroups 1 2, 5, 6, 7 2 5, 6, 7 5 7

The raw data were tabulated within each time interval, and the mean andstandard deviation were calculated for each endpoint and group. Forserum creatinine concentrations, treatment groups were compared to thereference groups using Repeated Measures Analysis of Covariance(RMANCOVA).

Repeated Measures Analysis of Covariance (RMANCOVA)

For endpoints measured at three or more post-test time intervals, arepeated measures analysis (mixed model) was conducted. For eachendpoint, the model tested for the effects of treatment, time, and theinteraction of treatment and time. Pre-test data (last measurementbefore dosing) were included in the model as a covariate.

If there was no significant (p>0.05) treatment by time interaction, thetreatment main effect was evaluated. If the treatment effect was notsignificant (p>0.05), the results were deemed not significant and nofurther analyses was conducted on the variable. If the treatment effectwas significant (p<0.05), linear contrasts were constructed for pairwisecomparison of each treatment group with the reference group. If theinteraction was significant (p<0.05), each treatment group was comparedto the appropriate reference group through the simple effect of‘treatment’ for each time point. These simple effect pairwisecomparisons were obtained from the ‘treatment by time’ interaction.

Results of all pair-wise comparisons are reported at the 0.05 and 0.01significance levels. All tests were two-tailed tests.

Results Serum Creatinine

As shown in FIG. 4, there were mild to moderate elevations creatinine atall intervals, relative to pre-test values. Creatinine tended tomaximally increase at 24 hours post-reperfusion, and then graduallydecrease over subsequent intervals. Changes in creatinine wereconsistent with reduced glomerular filtration secondary toprocedure-related renal injury (data not shown). In most treatmentgroups, and at most intervals, administration of the test article tendedto attenuate procedure-related elevations in creatinine, showingprotective effect of LVL-RecAP.

As illustrated in FIG. 5 there were dose-dependent elevations inalkaline phosphatase (ALP) activity in all treatment groups receivingthe test article at 24 hours post-reperfusion, relative to pretestvalues. ALP activity gradually decreased in treatment groups receiving≤1.6 mg/kg, but continued to progressively increase in animals receiving0.32 mg/kg/day.

Example 7 Safety Testing in Human Material and Methods Objectives

To evaluate the safety and tolerability of single and multiple doses ofrecombinant improved alkaline phosphatase (LVL-recAP) administered byintravenous (i.v.) infusion in healthy subjects.

To determine the pharmacokinetics (PK) of LVL-recAP after i.v. infusionof single and multiple doses of LVL-recAP in healthy subjects.

Design and Treatments

This is a 2-part, single-center study in a planned number of 50 healthysubjects. Part A will be a randomized, double-blinded,placebo-controlled, single ascending dose (SAD) study in up to 4sequential groups of 8 healthy male and female subjects each (6 onLVL-recAP and 2 on placebo). An attempt will be made to include in eachtreatment group an equal number of male and female subjects, with aminimal of 2 and a maximum of 4 females per group. A single dose ofLVL-recAP or placebo will be administered by a 1-hour i.v. infusion.Part B will be a randomized, double-blinded, placebo controlled,multiple ascending dose (MAD) study in up to 2 groups of 9 healthy maleand female subjects each (6 on LVL-recAP and 3 on placebo). An attemptwill be made to include in each treatment group an equal number of maleand female subjects, with a minimal of 2 and a maximum of 4 females pergroup. Subjects will receive a 1-hour i.v. infusion of LVL-recAP orplacebo on Days 1, 3 and 5. The following treatments will beadministered:

Part A

Group 1: 1-hour infusion of 200 U/kg LVL-recAPGroup 2: 1-hour infusion of 500 U/kg LVL-recAPGroup 3: 1-hour infusion of 1000 U/kg LVL-recAPGroup 4: 1-hour infusion of 2000 U/kg LVL-recAP

Part B

Group 5: 1-hour infusions of 500 U/kg LVL-recAP on Days 1, 2 and 3Group 6: 1-hour infusions of 1000 U/kg LVL-recAP on Days 1, 2 and 3

After completion of Day 9 of Group 1 and Day 4 of Group 2 of Part A, aninterim PK evaluation will be performed.

Depending of the results, infusion and PK sampling schedules may beadjusted for the remaining SAD and MAD groups.

In this first-in-human study, the subjects participating in the lowestdose level in Part A (Group 1) will be dosed according to a sentineldosing design to ensure minimal risk. This means that initially 2subjects will be dosed. One of these subjects will receive the activemedication LVL-recAP and the other subject will receive placebo. If thesafety and tolerability results of the first 24 hours following dosingfor the initial subjects are acceptable to the Principal Investigator,the other 6 subjects of the lowest dose level will be dosed in a placebocontrolled randomized manner (5 active and 1 placebo).

Observation Period

Part A: from Day −1 until 48 hours (Day 3) after drug administration.Short ambulant visits to the clinical research centre on Days 4, 6, 9and 15Part B: from Day −1 until 48 hours (Day 7) after last drugadministration. Short ambulant visits to the clinical research centre onDays 8, 10, 13 and 19 Subjects will be screened for eligibility within 3weeks prior to the (first) drug administration of each group of thestudy.

Follow-up examinations will take place on Day 15 (Part A) and Day 19(Part B).

Subjects

Part A: 32 healthy male and female subjectsPart B: 18 healthy male and female subjectsMain criteria for inclusionGender: male or femaleAge: 18-55 years, inclusiveBody Mass Index (BMI): 18.0-30.0 kg/m2, inclusive

Study Drug Active Drug

Active substance: LVL-recAP, an improved recombinant form of endogenoushuman alkaline phosphatase (AP)Activity: a hydrolase enzyme responsible for dephosphorylation ofmono-esters of phosphoric acidIndication: Acute kidney injury

Strength: 600, 1500, 3000 and 6000 U/mL

Dosage form: i.v. infusionManufacturer: pharmacy of PRA

Placebo

Substance: 20 mM citrate, 250 mM sorbitol, 2 mM MgCl2, 50μM ZnCl2, pH7.0Activity: noneIndication: not applicableStrength: not applicableDosage form: i.v. infusion

Manufacturer: Nova Laboratories Ltd, Gloucester Crescent, Wigston,Leicester, LE18 4YL, UK Criteria for Evaluation

Safety: adverse events (AEs), vital signs (including supine systolic anddiastolic blood pressure, pulse, body temperature, respiratory rate),12-lead electrocardiogram (ECG), continuous cardiac monitoring(telemetry), clinical laboratory (including clinical chemistry [AP isconsidered a PK parameter], hematology and urinalysis) tests, physicalexamination and anti-drug antibodies (ADA)Pharmacokinetics PK parameters based on analysis of serum concentrationsof LVL-recAP and AP activity.

Results

The dosing and observation time periods for all dosing groups have beenconcluded and no serious adverse events were observed in any of thegroups. The analyses of all parameters measured is ongoing.

Example 8 Materials and Methods Mice

The generation and characterization of the Alp1^(−/−) mice has beenreported previously (Narisawa et al., 1997). Alp1^(−/−) mice phenocopyinfantile HPP, including global deficiency of TNAP, PP, accumulation andmineralization defects (Fedde et al., 1999; Narisawa et al., 2001;Anderson et al., 2004; Millan et al., 2008). Dietary supplementationwith vitamin B6 briefly suppresses seizures and extends lifespan untilpostnatal days 18-22 but hypomineralization and accumulation of osteoidcontinue to worsen with age (Narisawa et al., 1997; Fedde et al., 1999;Narisawa et al., 2001; Millán et al., 2008). Therefore, all animals(breeders, nursing mothers, pups, and weanlings) in this study weregiven free access to modified laboratory rodent diet 5001 containingincreased levels (325 ppm) of pyridoxine. Genotyping was performed byPCR on genomic DNA as previously described (Yadav et al., 2011). TheInstitutional Animal Care and Use Committee (IACUC) approved all animalstudies.

Soluble Chimeric Human Alkaline Phosphatase (LVL-RecAP)

A solution of LVL-RecAP at 10.1 mg/ml in 25% glycerol w/v, 5 mMTris/HCl, 2 mM MgCl₂, 50 μM ZnCl₂, and at pH 8.0 was used. The enzymehad a purity of >99.99% as determined by high-pressure liquidchromatography.

Dose Response Study with LVL-RecAP

Alp1^(−/−) mice were divided into 5 cohorts: Vehicle-treated: Alp1^(−/−)mice treated with vehicle (n=14) only; LVL-RecAP1: Alp1^(−/−) micetreated with LVL-RecAP at 1 mg/kg/day (n=14); LVL-RecAP8: Alp1^(−/−)mice treated with LVL-RecAP at 8 mg/kg/day (n=12); and LVL-RecAP16:Alp1^(−/−) mice treated with LVL-RecAP at 16 mg/kg/day (n=10). Wild-typelittermates of Alp1^(−/−) mice served as reference animals and did notreceive injections (n=14). The vehicle or LVL-RecAP cohorts wereinjected daily SC into the scapular region. Injections were administeredbetween 8:00 and 11:00 AM. Volumes administered were calculated based onbody weight measured prior to injection. All treatments began onpostnatal day 1, and were repeated daily for up to 53 days or until thetime of necropsy.

Sample Collection

Necropsy was performed on postnatal day 53 (p53), 24 h after the finalinjection of LVL-RecAP for those animals that completed the experimentalprotocol, or sooner for those animals that appeared terminally ill.Avertin was administered intraperitoneally prior to euthanasia. Bloodwas collected into lithium heparin tubes by cardiac puncture. Necropsyconsisted of a gross pathology examination and x-rays.

Radiography and Microcomputed Tomography (μCT)

Radiographic images of skeletons were obtained with a Faxitron MX-20 DC4(Chicago, Ill., USA), using an energy of 20 kV. Hemi-mandibles werescanned at 30 kV. Whole dissected skulls from P21 mice were fixed, thenscanned at an 18 μm isotropic voxel resolution using the eXplore LocusSP μCT imaging system (GE Healthcare Pre-Clinical Imaging, London, ON,Canada). Measurements were taken at an operating voltage of 80 kV and 80mA of current, with an exposure time of 1600 ms using the Parker methodscan technique, which rotates the sample 180 degrees plus a fan angle of20 degrees. Scans were calibrated to a hydroxyapatite phantom and 3Dimages were reconstructed at an effective voxel size of 18 μm³. A fixedthreshold of 1400 Hounsfield Units was used to discriminate mineralizedtissue. Regions of interest (ROI's) for parietal and frontal bones wereestablished as 1 mm in length, 1 mm in width, depth equivalent tothickness of bone and position starting at a 0.75 mm distance fromsagittal and coronal sutures, as previously described (Liu et al.,2014). Parameters of bone volume, density and structure were measuredusing Microview version 2.2 software (GE Healthcare Pre-ClinicalImaging, London, ON) and established algorithms (Meganck et al., 2009;Umoh et al., 2009). Student's t-tests comparing quantitative resultswere performed to establish statistically significant differencesbetween genotypes. μCT bone data were analyzed and are reported inaccordance with the recommendations of Bouxsein et al. 2010 (Bouxsein etal., 2010). For dental imaging, dissected hemi-mandibles were scanned ona Scanco Medical μCT 50 (Scanco Medical AG, Brüttisellen, Switzerland)at 10 μm voxel size. Mandible z-stacks were exported as DICOM files andreoriented using ImageJ software (1.48r), with comparable coronal,sagittal, and transverse planes of section chosen for comparison. Forquantitative analysis, mandibles were scanned on a Scanco Medical μCT 35at 6 μm voxel size, 55KVp, 145 mA, with 0.36 degrees rotation step (180degrees angular range) and a 400 ms exposure per view. Scanco μCTsoftware (HP, DECwindows Motif 1.6) was used for 3D reconstruction andimage viewing. After 3D reconstruction, crown, enamel, roots, andalveolar bone volumes were segmented using global threshold 0.6 g/cc.Total volume (TV), bone (mineralized tissue) volume (BV), and tissuemineral density (TMD) were measured for the whole crown and separatelyfor enamel, root dentin, and alveolar bone in the furcation region. Forenamel and root, thickness was also measured.

Histological Analyses

Bone samples were cleaned, fixed in 4% paraformaldehyde/phosphatebuffered saline for 3 days at 4° C., and then transferred to 70% ethanolfor storage at 4° C. Plastic sections were prepared as describedpreviously (Yadav et al., 2012). Von Kossa and Van Gieson trichromestaining was performed on plastic sections as described previously(Narisawa et al., 1997). Von Kossa or Van Gieson-stained sections werescanned by ScanScopeXT system (Aperio, Vista, Calif., USA), and imageswere analyzed by using the Bioquant Osteo software (BioquantOsteoanalysis Co., Nashville, Tenn., USA). Left hemi-mandibles used forhistology were fixed in Bouin's solution for 24 h, and thendemineralized in AFS solution (acetic acid, formaldehyde, sodiumchloride), and embedded in paraffin for serial sectioning, as describedpreviously (Foster, 2012). For picrosirius red staining, deparaffinizedtissue sections were stained with 0.2% aqueous solution ofphosphomolybdic acid hydrate, 0.4% Direct red 80, and 1.3%2,4,6-trinitrophenol (Polysciences, Inc., Warrington, Pa.), as describedpreviously (Foster, 2012). Picrosirius red-stained sections wereobserved under polarized light for photomicrography.

Biomechanical Testing

After removal of muscle tissue, lengths of the femur, tibia, humerus andradius were measured with a caliper. Bones were frozen, wrapped in gauzecontaining a saline solution to avoid dehydration. Isolated femurs andtibias were assessed with three-point bending test by using the Instron1101 universal material testing machine as described previously (Huesaet al., 2011). Bones were slowly thawed and held at room temperatureprior to testing. The intact femurs and tibias were placed in thetesting machine on two supports separated by a distance of 15 mm andload was applied to the middle of the diaphysis, thus creating athree-point bending test at a speed of 2 mm min⁻¹.

PP_(i) Assay

PP_(i) concentrations in plasma were determined by differentialadsorption on activated charcoal of UDP-D[6-3H]glucose (AmershamPharmacia) from the reaction product 6-phospho [6-3H]gluconate, asdescribed (Hessle et al., 2002; Yadav et al., 2014).

Statistics

Considering that the different concentrations of LVL-RecAP resulted indifferent survival rates, it was not possible to compare the threetreatment cohorts in an age-matched fashion. For this reason, Student tunpaired, parametric, two-tailed test was performed to compare thetreated Alp1^(−/−) mice to the WT cohort. Differences were consideredsignificant when p<0.05. In order to compare the differences in thesurvival curves among the treatment cohorts, the Gehan-Breslow-Wilcoxontest was performed.

Results Increased Survival and Body Weight in LVL-RecAP-TreatedAlp1^(−/−) Mice

Survival in mice receiving 8 mg/kg/day (LVL-RecAP8) or 16 mg/kg/day(LVL-RecAP16) of LVL-RecAP was significantly improved compared to thevehicle-treated and the 1 mg/kg/day (LVL-RecAP1) cohort (p=0.001) (FIG.6A). Differences were statistically significant when the survival curvesof the treated cohorts were compared against each other (p<0.0001 forall comparisons). Median survival was 44, 22 and 19 days in theLVL-RecAP8, LVL-RecAP1 and vehicle-treated cohorts, respectively. Nomedian survival could be calculated for the LVL-RecAP16 cohort as theanimals lived until the termination of the experiment at day 53.Alp1^(−/−) animals weigh less than their WT littermates, starting aroundp7. Treatment with 1 mg/kg/day LVL-RecAP led to a statisticallysignificant increase in body weight compared to vehicle-treated mice,and a non-significant difference compared to WT littermates at 18 daysof treatment (FIG. 6B). Alp1^(−/−) mice usually die by p18-24 on VitaminB6 diet, therefore comparison of longer-lived LVL-RecAP8 and LVL-RecAP16cohorts to vehicle-treated mice was not possible, and these were insteadcompared to WT mice. Animals in the LVL-RecAP8 cohort weighedsignificantly less than their WT littermates from p18 (FIG. 6C) whilemice in the LVL-RecAP16 cohort showed normalization in body weight, andwere undistinguishable from WT mice at p53 (FIG. 6D).

LVL-RecAP Treatment Improves the Skeletal Phenotype of Alp1^(−/−) Mice

Radiographs of untreated Alp1^(−/−) mice (FIG. 7A) demonstrated profoundskeletal abnormalities including reduced tissue mineral density andfractured bones, as previously described (Yadav et al., 2011). We foundimprovement in the skeletal pathology in Alp1^(−/−) mice receiving 1mg/kg/day LVL-RecAP for 18 days (FIG. 7A), and the benefit was moreprofound in the LVL-RecAP8 and LVL-RecAP16 cohorts (FIG. 7B). The distalextremities displayed a normalized morphology, regardless of dose, whilepartial correction was observed in the spine, forelimbs, hindlimbs, andribcage for all treatment cohorts. LVL-RecAP8 and LVL-RecAP16 mice alsopresented minor cracks or fractures in femora and tibiae. The articularcontour in knees and elbows presented irregularities in LVL-RecAP8 andLVL-RecAP16 at p44 and p53, respectively (FIG. 7B).

To assess the degree of improvement of osteomalacia, we performed ahistomorphometric analysis of plastic-embedded undecalcified sections ofhindlimbs of LVL-RecAP-treated and WT control mice (FIGS. 8A and 9A). Wemeasured bone and osteoid volumes (FIGS. 8B, 8C) and also analyzed serumPP_(i) levels (FIGS. 9B, 9C). Consistent with earlier findings (Yadav etal., 2011), von Kossa staining revealed that Alp1^(−/−) mice show severedefects in mineralization, thin cortical bone, reduced trabecular boneand impaired ossification centers (FIG. 8A), whereas both LVL-RecAP8 andLVL-RecAP16-treated animals showed significantly enhanced cortical boneand improved secondary ossification centers. Histomorphometry (FIGS. 8B,8C) revealed that the BV/TV ratio (FIG. 8B), both in the LVL-RecAP8 andLVL-RecAP16 cohorts, was still significantly lower than in theage-matched WT controls (0.0321 and 0.0302, N=9), whereas the OV/BVpercentage (FIG. 8C) was significantly higher for both of the treatmentcohorts compared to WT controls (0.0001 and 0.0175, N=9). Differencesbetween LVL-RecAP8 and LVL-RecAP16 treatment cohorts were notstatistically significant. Histological trichrome staining (FIG. 9A) onplastic sections of hindlimbs confirmed these findings. Alp1^(−/−) micehave severely reduced mineralized bone mass (green-stained regions),with deficiencies in cortical and trabecular bone marked by osteoidaccumulation (red-stained regions). In contrast, 53-day-old WT controlshave robust cortical bones, trabecular bone present in the secondaryossifications and little osteoid at bone surfaces. Alp1^(−/−) mice inthe LVL-RecAP8 and LVL-RecAP16 cohorts display a significant improvementin cortical bone, especially in the LVL-RecAP16 cohort, where no majordifferences were noted compared to WT mice. While trabecular boneformation in the secondary ossification centers in Alp1^(−/−) mice isimproved by treatment, LVL-RecAP8 and LVL-RecAP16 mice still retainedlarger than normal regions of osteoid (red). In agreement with theskeletal findings, we found correction of the serum PP_(i)concentrations in all treatment cohorts. LVL-RecAP1 animals (FIG. 9B)harbor significantly reduced PP_(i) levels when compared to thevehicle-treated control animals. LVL-RecAP16 treated animals have PP_(i)levels that are statistically undistinguishable from that of WTlittermates.

Absence of Craniofacial Abnormalities in LVL-RecAP-Treated Alp1^(−/−)Mice

Alp1^(−/−) mice feature craniofacial shape abnormalities and coronalsuture fusion (Liu et al., 2014). To determine the extent to which thecraniofacial skeleton is affected by treatment in Alp1^(−/−) mice, weperformed μCT-based analyses of frontal and parietal cranial bones.Results at p21 show that both frontal and parietal bones of vehicletreated Alp1^(−/−) mice were significantly reduced in bone volumefraction, bone mineral content, bone mineral density, tissue mineralcontent and tissue mineral density when compared to WT or to treatedAlp1^(−/−) mice (Table 15). In contrast, neither frontal nor parietalbones of treated Alp1^(−/−) mice were significantly different from thoseof wild type mice. Adult skulls of LVL-RecAP16-treated Alp1^(−/−) micedid not appear different from WT in terms of size and shape (FIG.10A-10F).

TABLE 15 μCT analyses of cranial bones. Frontal and parietal bones wereanalzyed in WT, untreated Alpl^(−/−) (vehicle), and RecAP16-treatedAlpl^(−/−) mice at p21. Values are reported as means ± SD. Bone BoneTissue Tissue Bone Mineral Mineral Mineral Mineral Volume ContentDensity Content Density Fraction (mg) (mg/cc) (mg) (mg/cc) FRONTALAlpl^(−/−)  0.41 ± 0.06 *  0.008 ± 0.001 *  464 ± 27 *  0.004 ± 0.001 * 595 ± 28 * LVL- 0.72 ± 0.15 0.018 ± 0.005 615 ± 84 0.015 ± 0.006 697 ±57 RecAP16 WT 0.70 ± 0.12 0.019 ± 0.008 615 ± 79 0.016 ± 0.008 709 ± 56PARIETAL Alpl^(−/−)  0.52 ± 0.05 *  0.018 ± 0.001 *  494 ± 21 *  0.005 ±0.001 *  597 ± 12 * LVL- 0.72 ± 0.08 0.018 ± 0.007 608 ± 74 0.015 ±0.006 691 ± 61 RecAP16 WT 0.76 ± 0.10 0.020 ± 0.007 645 ± 73 0.017 ±0.007 726 ± 51 * Indicates statistical significance between genotypesand between treatment cohorts.

LVL-RecAP Treatment Partially Rescues Alp1^(−/−) Dentoalveolar Defects

Ablation of Alp1 in mice results in developmental mineralization defectsin cementum, dentin, alveolar bone, and enamel (Foster et al., 2014a;Foster et al., 2014b; McKee et al. 2011; Yadav et al., 2012), consistentwith case reports on human subjects with HPP. Absence of acellularcementum results in loss of periodontal attachment to the tooth rootsurface and premature tooth exfoliation, a hallmark of HPP.LVL-RecAP8-treated and vehicle-treated Alp1^(−/−) mice were compared toWT at p25-26, when molar tooth formation is near completion. Radiographyand μCT imaging revealed that, compared to controls (FIGS. 11A, 11D),untreated Alp1^(−/−) mouse mandibles featured grossly hypomineralizedbone, short molars with thin dentin and wide pulp chambers, and severelydefective incisors (FIG. 11B, 11F). By histology, Alp1^(−/−) mouse teethfeatured no acellular cementum, and alveolar bone osteoid invaded theperiodontal ligament (PDL) space, leading to ankylosis and loss of afunctional periodontium (FIG. 11G vs. 11E). Administration of 8mg/kg/day LVL-RecAP improved radiographic appearance of molar height,dentin thickness, and bone mineralization at p26, though the incisorremained defective (FIG. 11C, 11H). Histologically, though this dose ofLVL-RecAP did not restore acellular cementum to the root surface, themolar-associated PDL space and alveolar bone borders were bettermaintained (FIG. 11I).

The LVL-RecAP16 cohort was compared with WT at p50-53 to determineeffects on mature tooth structure and function. Radiography and μCTimaging indicated reduced alveolar and interproximal bone mineralizationaround molar teeth in Alp1^(−/−) mouse mandibles, compared to controls(FIG. 11J-11M). Molar tooth form and dentin appeared largely normalizedin LVL-RecAP16 mice, and this was confirmed by μCT analysis of the firstmolar (Table 16). Enamel in the LVL-RecAP16 group was not different fromWT in BV/TV or TMD. Molar crowns and roots showed mild but significantdecreases of 4-10% in BV/TV and TMD compared to WT, indicating dentinmineralization as not fully rescued, and root thickness was decreased by15%. Alveolar bone mineralization remained more severely defective inthe LVL-RecAP16 group, with BV/TV decreased by 27% and TMD decreased by13%, compared to WT. Incisor teeth in treated Alp1^(−/−) mice alsoremained severely affected (FIG. 11K, 11M).

By histology, LVL-RecAP16 mice featured a mix of mineralized alveolarbone and osteoid, and a reduced but maintained PDL space (FIG. 11N,11P). No tooth loss was noted in the LVL-RecAP16 group by p53, thoughperiodontal attachment remained deficient, as evidenced by lack ofcementum, PDL disorganization and detachment from the root surface, anddown growth of the junctional epithelium. However, small regions of PDLattachment to the tooth were noted in association with organized PDLfibers (FIG. 11O, 11Q), indicating the presence of some compromisedattachment that may function to retain molars.

TABLE 16 μCT analyses of dentoalveolar tissues. First mandibular molarsand associated alveolar bone were compared at p50-53 in WT (n = 5) andAlpl^(−/−) mice treated with RecAP16 (n = 4). Values are reported asmeans ± SD. TV BV BV/TV TMD Thickness (mm³) (mm³) (%) (g HA/cm³) (μm)Enamel WT 0.25 ± 0.03 0.24 ± 0.02  98.77 ± 0.52 1.69 ± 0.05 70.2 ± 0.5LVL-  0.20 ± 0.02 * 0.20 ± 0.02 * 98.97 ± 0.27 1.68 ± 0.03 63.0 ± 0.5RecAP16 Crown WT 0.61 ± 0.04 0.56 ± 0.03  91.06 ± 0.64 ND ND LVL-  0.53± 0.03 * 0.46 ± 0.03 *  86.92 ± 0.69 * ND ND RecAP16 Root WT 0.65 ± 0.050.56 ± 0.05  85.84 ± 0.74 1.07 ± 0.01 140.4 ± 8.4  LVP- 0.56 ± 0.06 0.44± 0.04 *  77.27 ± 2.37 *  1.02 ± 0.01 *  121.5 ± 1.7 * RecAP16 AlveolarBone WT 0.44 ± 0.04 0.29 ± 0.05  65.72 ± 5.08 1.01 ± 0.02 ND LVL-  0.32± 0.05* 0.15 ± 0.02 *  47.91 ± 4.25 *  0.88 ± 0.02 * ND RecAP16 * p <0.05 by independent samples t-test ND = Not determined

Literature References to Example 8

-   Anderson H. C., Sipe J. B., Hessle L., Dhanyamraju R., Atti E.,    Camacho N. P., Millán J. L. Impaired calcification around matrix    vesicles of growth plate and bone in alkaline phosphatase-deficient    mice. Am. J. Pathol. 2004; 164(3):841-847.-   Bouxsein M L, Boyd S K, Christiansen B A, Guldberg R E, Jepsen K J    and Müller R. Guidelines for assessment of bone microstructure in    rodents using micro-computed tomography. J Bone Miner Res 2010;    25(7):1468-86.-   Fedde K N, Blair L, Silverstein, J, Coburn S P, Ryan L M, Weinstein    R S, Waymire K, Narisawa S, Millán, J L, MacGregor GR, Whyte M P,    Alkaline phosphatase knockout mice recapitulate the metabolic and    skeletal defects of infantile hypophosphatasia. J Bone Miner Res    1999; 14: 2015-2026.-   Foster B L, Nagatomo K J, Nociti F H, Fong H, Dunn D, Tran A B, Wang    W, Narisawa S, Millán J L, Somerman M J 2012 Central role of    pyrophosphate in acellular cementum formation. PLoS One 7(6):e38393.-   Foster B. L., Nagatomo K. J., Tso H. W., Tran A. B., Nociti F. H.,    Jr., Narisawa S., Yadav M. C., McKee M. D., Millan J. I.,    Somerman M. J. Tooth root dentin mineralization defects in a mouse    model of hypophosphatasia. J Bone Miner Res. 2013; 28(2):271-82.    Foster B L, Nociti F H, Jr., Somerman M J (2014a). The rachitic    tooth. Endocr Rev 35(1):1-34.-   Foster B L, Ramnitz M S, Gafni R I, Burke A B, Boyce A M, Lee J S et    al. (2014b). Rare Bone Diseases and Their Dental, Oral, and    Craniofacial Manifestations. J Dent Res 93(7 suppl).7S-19S.-   Hessle L., Johnson K. A., Anderson H. C., Narisawa S., Sali A.,    Goding J. W., Terkeltaub R., Millán J. L. Tissue-nonspecific    alkaline phosphatase and plasma cell membrane glycoprotein-1 are    central antagonistic regulators of bone mineralization. Proc. Natl.    Acad. Sci. U.S.A. 2002; 99(14):9445-9449.-   Huesa C., Yadav M. C., Finnilá M. A., Goodyear S. R., Robins S. P.,    Tanner K. E., Aspden R. M., Millán J. L., Farquharson C. PHOSPHO1 is    essential for mechanically competent mineralization and the    avoidance of spontaneous fractures. Bone. 2011; 48(5):1066-1074.-   Liu J, Nam H K, Campbell C, Gasque K C, Millán J L, Hatch N E.    Tissue-nonspecific alkaline phosphatase deficiency causes abnormal    craniofacial bone development in the Alp1(−/−) mouse model of    infantile hypophosphatasia. Bone 2014; 67:81-94.-   McKee M. D., Nakano Y., Masica D. L., Gray J. J., Lemire I., Heft    R., Whyte M. P., Crine P., Millán J. L. Enzyme replacement therapy    prevents dental defects in a model of hypophosphatasia. J. Dent.    Res. 2011; 90(4):470-476.-   Meganck J A, Kozloff K M, Thornton M M, Broski S M and Goldstein    S A. Beam hardening artifacts in micro-computed tomography scanning    can be reduced by X-ray beam filtration and the resulting images can    be used to accurately measure BMD. Bone 2009; 45(6):1104-1116.-   Millán J L, Narisawa S, Lemire I, Loisel T P, Boileau G, Leonard P,    Gramatikova S, Terkeltaub R, Pleshko Camacho N, McKee M D, Crine P    and Whyte M P, Enzyme replacement therapy for murine    hypophosphatasia. J Bone Miner Res 2008; 23: 777-787.-   Narisawa S, Wennberg C. Millán J L, Abnormal vitamin B6 metabolism    in alkaline phosphatase knock-out mice causes multiple    abnormalities, but not the impaired bone mineralization. J Pathol    2001; 193: 125-133.-   Narisawa S, Fröhlander N, Millán J L, Inactivation of two mouse    alkaline phosphatase genes and establishment of a model of infantile    hypophosphatasia. Dev Dyn 1997; 208: 432-446.-   Umoh J U, Sampaio A V, Welch I, Pitelka V, Goldberg H A, Underhill T    M et al. In vivo micro-CT analysis of bone remodeling in a rat    calvarial defect model. Phys Med Biol 2009; 54(7):2147-61.-   Yadav M. C., Lemire I., Leonard P., Boileau G., Blond L., Beliveau    M., Cory E., Sah R. L., Whyte M. P., Crine P., Millán J. L. Dose    response of bone-targeted enzyme replacement for murine    hypophosphatasia. Bone. 2011; 49(2):250-256.-   Yadav M. C., de Oliveira R. C., Foster B. L., Fong H., Cory E.,    Narisawa S., Sah R. L., Somerman M., Whyte M. P., Millán J. L.    Enzyme replacement prevents enamel defects in hypophosphatasia    mice. J. Bone Miner. Res. 2012; 27(8):1722-1734.-   Yadav, M. C., Huesa, C., Narisawa, S., Hoylaerts, M. F., Moreau, A.,    Farquharson, C. and Millán, J. L. Ablation of osteopontin improves    the skeletal phenotype of Phospho1^(−/−) mice. J. Bone Miner. Res.    In Press (2014).

Example 9 Alkaline Phosphatase Protects Against Renal InflammationMethods Cell Culture

Routinely, ciPTEC were cultured at 33° C. (Wilmer, 2010). Cells weretransfected with Simian Virus 40 T-antigen and the essential catalyticsubunit of human telomerase, allowing them to constantly proliferate.Cells were cultured in DMEM/Ham's F-12, phenolred-free (Gibco, Paisly,United Kingdom), supplemented with ITS (5 μg/ml insulin, 5 μg/mltransferrin, 5 ng/ml selenium; Sigma-Aldrich, Zwijndrecht, TheNetherlands), 36 ng/ml hydrocortisone (Sig-ma-Aldrich), 10 ng/mlepidermal growth factor (Sigma-Aldrich), 40 μg/ml tri-iodothyronine and10% fetal calf serum (Greiner Bio-One, Kremsmünster, Austria). Precedingan experiment, cells were seeded in a well-plate (48400 cells/cm²),incubated for 1 day at 33° C. followed by a 7-day maturation period at37° C. On the day of the experiment, cells were pre-incubated for twohrs with LVL-RecAP (1, 5 or 10 U/ml, kind gift from AM-Pharma, Bunnik,The Netherlands) (Kiffer-Moreira, 2014), followed by incubation for 24hrs with 10 μg/ml LPS (E. coli 0127:B8; Sigma-Aldrich) dissolved in 10mM HEPES HBSS, pH 7.4 (HEPES: Roche Diagnostics, Mannheim, Germany;HBSS: Gibco). Alternatively, 10 U/ml LVL-RecAP (˜17 μg/ml) wasadministered to LPS-incubated cells simultaneously or after two hrs.Control cells were incubated with culture medium solely. DLPS (E. coli055:B5; Sigma-Aldrich, 10 μg/ml) and inactive LVL-RecAP (17 μg/ml, kindgift from AM-Pharma) were used as negative controls. In different setsof experiments, LPS was substituted for human TNF-α recombinant protein(Ebioscience, Vienna, Austria), or supernatant of PBMCs, prestimulatedfor 24 hrs with or without LPS (1 ng/ml). All experiments (n=5) wereminimally performed in duplicate.

Isolation of Peripheral Blood Mononuclear Cells

PBMCs were isolated from buffy coats obtained from healthy blood donors(blood bank Nijmegen, n=5) by differential centrifugation overFicoll-Pague Plus (GE Healthcare, Diegem, Belgium). PBMCs wereresuspended in RPMI-1640 medium (Gibco) enriched with 0.5 mg/mlgentamicin (Sigma-Aldrich), 1 mM pyruvate (Gibco) and 2 mM glutamax(Gibco). Cells were seeded in 96-well plates at a density of 0.5×10⁶cells/well, pre-incubated with or without AP (10 units/ml) for 2 hrs,followed by LPS incubation (1 ng/ml) for 24 hrs. All experiments wereperformed in duplicate.

ATP Measurement and Cell Viability Assay

Supernatant was collected 30 minutes after LPS administration, with orwithout LVL-RecAP-pretreatment, followed by direct measurement of ATPproduction using the ATP Bioluminescence Assay Kit CLS II (RocheDiagnostics) according to manufacturer's protocol. Cell viability wasassessed after 24 hrs of LPS incubation by performing the MTT assay. Inshort, medium was substituted for 100 μl prewarmed MTT-solution(Sigma-Aldrich; 0.5 mg/ml in culture medium), incubated for 3 hrs at 37°C., followed by the addition of 200 μl DMSO to solubilize intracellularprecipitated formazan crystals. Dye extinction was measured at 570 nmwith wavelength correction of 670 nm.

Animal Model

Animal experiments were performed according to the National Institutesof Health guidelines and protocols were approved by the institutionalreview board for animal experiments. Male specific-pathogen freeSprague-Dawley rats (RjHan:SD: Janvier, France) were divided into threegroups: placebo (n=6), LPS (n=6) or LPS+LVL-RecAP (n=6). A baselineplasma sample (lithium-heparin blood) was collected seven days precedingthe experiment through a tail vein puncture using a Multivette(Sarstedt, Etten-Leur, the Netherlands). Three days preceding theexperiment, the baseline renal function was assed as FITC-sinistrint_(1/2) (Schock-Kusch, 2011). At t=0 hrs, placebo (0.9% NaCl, saline) or0.3 mg/kg BW LPS (E. coli 0127:B8, dissolved in saline) was administeredas an IV bolus to induce LPS-induced renal failure. At t=1.5 hrs plasmawas obtained as described above. At t=2 hrs, rat received an IV bolus ofplacebo or LVL-RecAP (1000 U/kg BW, diluted in saline) followed by asecond measurement of renal function. At t=5 hrs, all animals received 5ml saline (s.c.) to prevent dehydration, followed by a 16 hour urinecollection period. At t=21.5 hrs the third transcutaneous measurementwas performed. At t=24 hrs, rats were anesthetized (i.p., 3 mg/kg BWxylazine and 80 mg/kg BW ketamine 10%), a retrobulbar lithium-heparinblood sample was withdrawn to obtain plasma, and whole body perfusionwas started (6 min, saline+50 IU/ml heparin, 210 mbar; 3 min, 4%paraformaldehyde (PFA; 210 mbar). After saline perfusion, the rightkidney was carefully removed, snap frozen and stored at −80° C. untilprocessing. The left kidney, removed after PFA perfusion, was stored in4% PFA at 4° C. until processed for histology and immunohistochemistry.One animal from the LPS+LVL-RecAP group and one urine sample from theplacebo group were excluded due to injection and collectiondifficulties, respectively.

Renal Function Measurements

Renal function was assessed in freely moving awake rats throughtranscutaneously measured elimination kinetics of FITC-sinistrin(Fresenius Kabi, Linz, Austria), a commercially available marker of GFR,by using a novel measurement device as published before (Schock-Kusch2011, Schock-Kusch 2009). Briefly, rats were anesthetized by isofluraneinhalation (5% induction, 1.5-2% maintenance; Abbott Laboratories, Ill.,USA) and shaved on the back. The optical part of the device was fixed onthis depilated region using a specifically designed double-sidedadhesive patch (Lohmann GmbH, Neuwied, Germany), whereas the electronicpart of the device was incorporated into a rodent jacket (LomirBiomedical, Malone, USA). After establishing the baseline signal,FITC-sinistrin (5 mg per 100 g BW, diluted in buffered saline) wasinjected in the tail vein. Thereafter, the animals were allowed torecover from anesthesia while the measurement continued forapproximately 120 minutes post-injection. T_(1/2) was calculated by aone-compartment model applied on the transcutaneously measuredFITC-sinistrin elimination kinetics (Schock-Kusch, 2009). In addition,parameters of renal function were determined in plasma and urine samplesusing the Hitachi 704 automatic analyzer (Boehringer Mannheim, Mannheim,Germany). Fractional urea excretion and endogenous creatinine clearancewere calculated with average plasma values of t=1.5 and t=24.

Histology and Immunohistochemistry

After fixation for at least 24 hrs, tissue was processed, embedded inparaplast and sectioned at 3 μM thickness. For routine histology, HEstaining was performed on renal tissue. Renal injury was assessed usinga scoring system with a scale from 0 to 4 (0=no changes; 4=severe damagee.g. marked tubule cell changes). KIM-1 was detected by primary antibodygoat-anti-rat KIM-1 (1:50; AF3689, R&D Systems, Abingdon, UK) andsecondary antibody rabbit-anti-goat IgG (1:200; P0449, DAKO, Heverlee,Belgium). Immunostaining was visualized with VECTASTAIN Eline ABC systemreagents (Vector Labs, Amsterdam, Netherlands) and 3,3′-Diaminobenzidine(DAB, Sigma-Aldrich), followed by haematoxyline counterstain. Allscoring was performed in a blinded fashion.

Cytokines and Renal Injury Markers

Human ELISA kits (R&D Systems) were used to determine TNF-α, IL-6 andIL-8 in supernatant according to manufacturer's instructions. Plasmacytokine levels (IL-1ß, IL-6, IL-10, TNF-α, INF-γ) were determined by asimultaneous Luminex assay according to the manufacturer's instructions(Millipore, Cork, Ireland). KIM-1 and NGAL were determined by ELISA (R&DSystems) according to manufacturer's instructions.

Tissue Homogenization

Snap frozen kidneys were homogenized by the TissueLyser LT (Qiagen,Venlo, The Netherlands) according to manufacturer's instructions, inTissue Protein Extraction Reagent (T-PER; Thermo Scientific, Rockford,USA), supplemented with complete EDTA-free protease inhibitor cocktailtablets (Roche Applied Science, Almere, The Netherlands). Total proteincontent was determined using the bicinchonicic acid protein assay kit(Thermo Scientific) and samples were stored at −80° C. until assayed.

Real-Time PCR Analyses

RNA was extracted from frozen cell pellet or pulverized kidneys (2000,30 sec; Mikro-dismembrator U, Sartorius Stedim Biotech, Aubagne Cedex,France) by Trizol reagent. RNA was reverse-transcribed into cDNA usingMoloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Invitrogen,Breda, The Netherlands). Real-time quantitative PCR (RQ-PCR) wasperformed using Taqman® (Applied Biosystems, Carlsbad, USA). Genes wereamplified and normalized to the expression of GAPDH (ciPTEC: Ct:18.9±0.1; renal tissue: Ct: 24.8±0.2). The PCR reaction started with a 2min incubation step at 50° C. followed by initial denaturation for 10min at 95° C., and 40 cycles of 15 sec at 95° C. and 1 min at 60° C.Differences between groups were calculated by the comparative ΔΔCtmethod. Primers/probe sets are summarized in Table 17.

Urinary Purine Content

Urinary adenosine, AMP, ADP, ATP and cAMP content was determined byHPLC. In brief, 4 volumes of urine were mixed with 1 volume ofchloroacetaldehyde (6× diluted in 1M acetate buffer, pH 4.5;Sigma-Aldrich), followed by derivatization (60 min, 70° C., 500 rpm) andcentrifugation (3 min, RT, 13400 rpm), whereafter the supernatant wastransferred to a HPLC vial and injected. Purines were separated by HPLCsystem (Thermo Scientific) using a Polaris C18-A column (150×4.6 mm)with gradient elution using eluent A (0.1M K₂HPO₄, 10 mM TBAHS (pH 6.5),and 2% MeOH) and eluent B (H₂O: ACN: THF; 50:49:1). Retention times were7.1 (adenosine), 8.4 (AMP), 12.5 (ADP), 16.2 (ATP) and 14.8 min (cAMP).Quantification was based on peak areas of the samples and referencestandards measured with fluorescence (excitation: 280 nm; emission: 420nm).

Statistical Analysis

Data are expressed as mean±SEM or median [25th percentile, 75thpercentile]. Normality of data was assessed by Kolmogorov-Smirnov test.Statistical differences between groups were estimated by ANOVA withpost-hoc comparisons using Bonferroni's multiple comparison test or byKruskal-Wallis test with Dunn's post-test. A two-sided p-value less than0.05 was considered statistically significant. All tests were performedwith Graphpad Prism 5.00 for Windows (Graphpad Software Inc. San Diego,Calif., USA).

LVL-RecAP Attenuates the LPS-Induced Inflammatory Response In Vitro

Pre-treatment of human ciPTEC (Wilmer, 2010) with LVL-RecAPdose-dependently attenuated the LPS-induced cytokine production ofTNF-α, IL-6 and IL-8 on the gene and protein level (FIG. 12A-12F).Detoxified LPS (dLPS) was used as a negative control and had no effect.Similar protective results at the protein level were obtained whenLVL-RecAP was administered simultaneously with LPS or 2 hrs after LPSexposure (FIG. 12G-12I). A control experiment was performed to verifywhether LVL-RecAP dephosphorylates the cytokines excreted in the medium,which was not the case (FIG. 17A-17C). To confirm that theLVL-RecAP-induced reduction in cytokine production was due to thedephosphorylating nature of the enzyme, the effect of inactive LVL-RecAPthat lacks hydrolyzing properties was investigated. Inactive LVL-RecAPdid not attenuate the LPS-induced inflammatory response in ciPTEC (FIG.12J-12L).

The In Vitro Effects of LVL-RecAP are Renal Specific and not Restrictedto LPS-Induced Inflammation

To investigate further the renal protective mechanism of LVL-RecAP,ciPTEC were incubated with the pro-inflammatory cytokine TNF-α, whichcannot be dephosphorylated by calf IAP (Chen, 2010). The TNF-α inducedcytokine production of IL-6 and IL-8 was also attenuated by LVL-RecAPpretreatment, whereas inactive LVL-RecAP had no effect (FIG. 13A-13B).

In the pathogenesis of sepsis-associated AKI, LPS induces a localinflammatory response through binding to TLR4 expressed on PTEC (ciPTECCt: 30.5±3.9; n=5). Another hallmark of the disease is the systemicinflammatory response, which affects both renal epithelial andendothelial cells causing the development of AKI (Peters, 2014). Tomimic this endotoxin-induced renal inflammation, ciPTEC were incubatedwith the supernatant of LPS-stimulated peripheral blood mononuclearcells (PBMCs, 1 ng/ml LPS). This induced the production of IL-6 andIL-8, which was decreased when ciPTEC were pretreated with LVL-RecAP(FIG. 13C-13D, TNF-α was not detectable). Together with the finding thatinflammatory responses mediated by TNF-α were attenuated by LVL-RecAPtreatment, this suggests the presence of another mediator targeted byLVL-RecAP. In contrast, pretreatment of PBMCs with LVL-RecAP did notaffect the LPS-induced inflammatory response in these cells (FIG.13E-13F), indicating that the effects of LVL-RecAP are kidney specific.

LVL-RecAP May Exert Renal Protective In Vitro Effects Through theATP/Adenosine Pathway

A second potential target of LVL-RecAP is ATP, released during cellstress caused by e.g. inflammation and hypoxia (Eltzschig, 2012).Extracellular ATP has detrimental effects, but can be converted byectonucleotidases (e.g. AP) into ADP, AMP and eventually into adenosine,exerting anti-inflammatory and tissue-protective effects through bindingto one of the adenosine receptors A1, A2A, A2B and A3 (Bauerle, 2011, DiSole, 2008). Interestingly, while the adenosine receptors A1, A2B and A3expression in ciPTEC were not affected by LPS incubation (data notshown), the A2A expression was up-regulated upon LPS stimulation (foldincrease: 4.1±0.4; p<0.001 compared to placebo). This effect wasattenuated by LVL-RecAP co-treatment (fold increase: 2.9±0.2; p<0.001compared to placebo; p<0.05 compared to LPS), suggesting a role of theadenosine pathway in the protective effect of LVL-RecAP. Furthermore, weobserved increased extracellular ATP concentrations following LPSincubation, which was more pronounced with a higher LPS concentrationbut reversed by LVL-RecAP preincubation (FIG. 14). LPS did not affectcell viability up to 24 hrs (data not shown). This supports thehypothesis that LVL-RecAP may exert its renal protective effect throughthe ATP/adenosine pathway.

LVL-RecAP Treatment During LPS-Induced AKI in Rats Attenuates ImpairedRenal Function

To confirm the beneficial effects of LVL-RecAP in vivo, AKI was inducedin rats by LPS (0.3 mg/kg BW) and renal function was assessed by thetranscutaneous measurement of the fluorescein isothiocyanate(FITC)-labeled sinistrin kinetics as previously reported (Schock-Kusch,2011). FITC-sinistrin is cleared by the kidneys through filtrationsolely and its disappearance from the plasma compartment can be measuredtranscutaneously in real-time (Schock-Kusch, 2011, Schock-Kusch, 2009).This allows investigating the progression of AKI in a more accuratemanner as compared to the commonly used creatinine clearance. PrecedingLPS injection, a baseline blood sample was drawn to determine clinicalparameters and plasma cytokines, and the baseline FITC-sinistrinhalf-life (t_(1/2)) was determined from the measured kinetics toascertain homogeneity between groups (data not shown). After 1.5 hrs,LPS treatment resulted in increased plasma cytokines levels,abnormalities in several plasma parameters (Table 18), piloerection,diarrhea and reduced spontaneous activity, confirming the presence ofsystemic inflammation. Two hrs after LPS administration, rats weretreated with LVL-RecAP (1000 U/kg BW) or placebo (saline), directlyfollowed by transcutaneous renal function measurements. LPSsignificantly prolonged FITC-sinistrin t_(1/2), revealing a significantreduction in renal function. This trend was attenuated by LVL-RecAPtreatment (FIG. 15A). In all groups, renal function was fully recoveredwithin 24 hrs (FIG. 15B). Still, plasma urea levels were significantlylower in LVL-RecAP treated animals compared to animals that received LPSwithout LVL-RecAP (Table 19). Also, LVL-RecAP treatment prevented theLPS-induced increase of fractional urea excretion (FIG. 15C) and theLPS-induced decrease of endogenous creatinine clearance (FIG. 15D).LVL-RecAP bio-activity was confirmed in plasma and showed an eightfoldincrease 22 hrs after injection (Placebo; 293±12 U/ml; LPS: 260±13 U/ml;LPS+AP: 2150±60 U/ml; p<0.0001).

LVL-RecAP Prevents Renal Injury During LPS-Induced AKI In Vivo

The renal protective effect of LVL-RecAP on LPS-induced AKI wasinvestigated further through evaluation of renal histology and specifictubular injury markers. No differences in histology were found betweenthe treatment groups, with changes that ranged from no damage (0) tillminimal degenerative changes like foamy appearance and minimal swellingof proximal tubular cells (1) and foamy appearance and moderate swellingas well as a few cases of apoptosis (2) (Placebo; 1 [0.75-2]; LPS: 1.5[0-2]; LPS+LVL-RecAP: 1 [0-1.0]). LPS treatment resulted in asignificant increase in renal IL-6 expression levels, while othercytokines and injury markers (MPO, myeloperoxidase; BAX, Bcl2-associatedX protein; iNOS, inducible nitric oxide synthase) were not affected(Table 20). LVL-RecAP could not reduce renal IL-6 expression levels, butdid enhance renal expression of the anti-inflammatory cytokine IL-10(Table 20). Furthermore, LPS administration resulted in a significantincrease in the urinary excretion of kidney injury molecule (KIM)-1 andneutrophil gelatinase-associated lipocalin (NGAL), which was accompaniedby increased renal gene expression levels. This effect was prevented byLVL-RecAP co-administration (FIG. 16A-16B, Table 20). Similar effects ofLVL-RecAP were observed for plasma NGAL levels (FIG. 16C) and for renalprotein levels of KIM-1 (FIG. 16D), which was localized primarily to theapical surface of proximal tubule epithelial cells (FIG. 16E).

Reduced Urinary Adenosine Excretion During LPS-Induced AKI In Vivo

In order to elucidate further the renal protective mechanism ofLVL-RecAP, we investigated the role of the ATP-adenosine pathway. LPStreatment tended to reduce the gene expression levels in the kidney forall four adenosine receptors, of which only adenosine receptor A3reached statistical significance (Table 20). Interestingly, LPStreatment significantly decreased the urinary excretion of adenosine(placebo: 68.0±7.8 pg adenosine/10 μg creatinine; LPS: 19.4±6.3 pgadenosine/10 μg creatinine; p<0.001), without altering the excretion ofcAMP, ATP, ADP and AMP (data not shown). This may suggest that thekidney utilizes adenosine during LPS-induced AKI. LVL-RecAP treatmenthad no effect on adenosine receptor gene expression (Table 20), or onurinary adenosine excretion (LPS+LVL-RecAP: 16.7±6.8 μg adenosine/10 μgcreatinine: p<0.001 compared to placebo) compared to LPS alone.

Tables

TABLE 17 Primer/probe specifications Gene symbol Gene name Assay IDciPTEC GAPDH glyceraldehyde-3-phosphate Hs02758991_g1 dehydrogenaseTNF-α tumor necrosis factor Hs01113624_g1 IL-6 interleukin 6Hs00985639_m1 IL-8 interleukin 8 Hs00174103_m1 TLR4 toll-like receptor 4Hs00152939_m1 ADORA1 adenosine A1 receptor Hs00379752_m1 ADORA2Aadenosine A2a receptor Hs00169123_m1 ADORA2B adenosine A2b receptorHs00386497_m1 ADORA3 adenosine A3 receptor Hs01560269_m1 Rat kidneyGAPDH glyceraldehyde-3-phosphate Rn01775763_g1 dehydrogenase IL-1βinterleukin 1 beta Rn00580432_m1 IL-6 interleukin 6 Rn01410330_m1 IL-10interleukin 10 Rn00563409_m1 TNF-α tumor necrosis factor Rn99999017_m1IFN-γ interferon gamma Rn00594078_m1 HAVCR1 hepatitis A virus cellularreceptor 1 Rn00597703_m1 LCN2 lipocalin 2 Rn00590612_m1 MPOmyeloperoxidase Rn01460204_m1 BAX Bcl2-associated X proteinRn02532082_g1 NOS2 nitric oxide synthase 2, inducible Rn00561646_m1ADORA1 adenosine A1 receptor Rn00567668_m1 ADORA2A adenosine A2areceptor Rn00583935_m1 ADORA2B adenosine A2b receptor Rn00567697_m1ADORA3 adenosine A3 receptor Rn00563680_m1

TABLE 18 Plasma cytokines and experimental parameters Placebo LPS LPS +recAP Cytokines IL-1β (pg/ml) 9 ± 9  6913 ± 1362^(#) 5764 ± 1259^(#)IL-6 (pg/ml) ND  74294 ± 11240^(#) 70247 ± 13812^(#) IL-10 (pg/ml) 17 ±17 10054 ± 2017^(#) 8693 ± 2513^(#) TNF-α (pg/ml) 1 ± 1 21071 ± 3375^(#)10514 ± 889^(#) *  INF-γ (pg/ml) ND ND ND Plasma parameters Creatinine0.16 ± 0.01  0.30 ± 0.05 0.25 ± 0.04  (mg/dl) Urea (mg/dl) 28 ± 2  39 ±2^(#)  48 ± 3^(#) * Lactate (mg/dl) 24 ± 5  34 ± 2  33 ± 3   Glucose 143± 3  260 ± 25^(#) 216 ± 13^(#)  (mg/dl) Protein 58 [56-58]  58 [48-61] 56 [56-58]  (mg/ml) Calcium 2.59 ± 0.03  2.43 ± 0.03^(#) 2.41 ± 0.06^(#)(mmol/l) Inorganic 3.01 ± 0.10  2.76 ± 0.09 2.81 ± 0.10  Phosphorus(mmol/l) Sodium 149 ± 2  148 ± 2   146 ± 3   (mmol/l) Potassium 5.89[5.66-6.21] 4.87 [4.66-5.23] 5.08 [4.76-5.18] (mmol/l) Plasma parameterswere determined 1.5 hrs after LPS administration. Data is expressed asmean ± SEM, and median [25^(th) percentile, 75^(th) percentile],depending on the distribution of each parameter. Differences indistribution of plasma parameters compared to t = 24 is likely to berelated to sample size. Significant differences estimated using one-wayANOVA with Bonferroni post-test, or Kruskal-Wallis test with Dunnspost-test. Placebo, LPS n = 6; LPS + recAP n = 5. ^(#)p < 0.05 comparedto placebo. * p < 0.05 compared to LPS. LPS, Lipopolysaccharide; recAP,recombinant Alkaline Phosphatase; ND: not detected.

TABLE 19 Plasma and urinary parameters Placebo LPS LPS + recAP Plasmaparameters Creatinine 0.20 ± 0.01 0.24 ± 0.02 0.20 ± 0.01 (mg/dl) Urea(mg/dl) 28 ± 2  48 ± 4^(#)   36 ± 2* Lactate (mg/dl) 10 [8-31] 14[12-21] 12.0 [10-14] Glucose 144 ± 7  132 ± 11  161 ± 3  (mg/dl) Protein56 [54-57] 56 [52-57] 54 [54-58] (mg/ml) Calcium 2.84 [2.7-3.1] 2.65[2.6-2.7] 2.70 [2.6-2.8] (mmol/l) Inorganic 2.95 ± 0.09 3.06 ± 0.10 2.95± 0.09 Phosphorus (mmol/l) Sodium 150 [148-157] 154 [147-155] 150[141-154] (mmol/l) Potassium 4.24 ± 0.04 4.32 ± 0.10 4.42 ± 0.24(mmol/l) Urinary parameters Creatinine (mg) 5.4 ± 0.3 6.1 ± 0.3 5.7 ±0.6 Urea (mg) 267 ± 26  482 ± 21^(#)   440 ± 19^(#)   Albumin (μg) 0[0-54] 0 [0-663] 310 [0-419] Glucose (mg) 1.59 ± 0.25 1.74 ± 0.28 0.99 ±0.27 Protein (μg) 38 ± 6  67 ± 9  61 ± 9  Calcium 4.1 ± 0.7 7.2 ± 0.97.4 ± 1.7 (μmol) Inorganic 0.50 [0.3-0.9] 0.63 [0.6-1.0] 0.64 [0.6-0.8]Phosphorus (mmol) Sodium 1.6 [1.3-2.0] 1.5 [1.3-1.7] 1.5 [1.4-2.4](mmol) Potassium 1.6 ± 0.3  2.3 ± 0.4^(#)  1.9 ± 0.1^(#) (mmol) Plasmaparameters were determined 24 hrs after LPS administration. Urinaryparameters were determined between 5 and 21 hrs after LPSadministration. Data is expressed as mean ± SEM, and median [25^(th)percentile, 75^(th) percentile], depending on the distribution of eachparameter. Significant differences estimated using Kruskal-Wallis testwith Dunns post-test or one-way ANOVA with Bonferroni post-test.Placebo, LPS n = 6; LPS + recAP n = 5; urinary parameters: Placebo n =5. ^(#)p < 0.05 compared to placebo. *p < 0.05 compared to LPS. LPS,Lipopolysaccharide; recAP, recombinant Alkaline Phosphatase.

TABLE 20 Renal gene expression levels Ct values Fold increase(2{circumflex over ( )}ΔΔCt) Placebo LPS LPS + recAP Placebo LPS LPS +recAP Cytokines IL-1β 27.7 ± 0.5 26.6 ± 0.2 26.9 ± 0.3 1.1 ± 0.2 1.6 ±0.5 1.7 ± 0.5 IL-6 37.4 [36.3-38.0] 33.5 [33.3-34.8] 34.7 [34.4-36.4]1.2 ± 0.3  4.9 ± 0.5^(#)  5.2 ± 1.2^(#) IL-10 34.8 ± 0.4 32.5 ± 0.1 32.9± 0.3 1.1 ± 0.2 3.3 ± 0.9  5.5 ± 1.2^(#) TNF-α 36.7 ± 0.2 35.8 ± 0.437.2 ± 0.5 1.1 ± 0.2 1.0 ± 0.1 1.0 ± 0.3 INF-γ 36.3 ± 0.3 35.3 ± 0.435.6 ± 0.2 1.1 ± 0.2 1.3 ± 0.3 1.7 ± 0.2 Injury markers KIM-1 31.7 ± 0.422.9 ± 0.7 25.3 ± 0.6 0.8 [0.6-1.9]   430 [195-530]^(#) 113 [43-336] NGAL 29.1 ± 0.2 24.1 ± 1.4 25.5 ± 1.6 1.2 ± 0.3 33 ± 9^(# ) 28 ± 8  MPO32.4 ± 0.3 31.4 ± 0.7 33.0 ± 1.0 1.0 [0.4-2.9] 1.4 [0.4-3.9] 0.6[0.3-3.2] BAX 25.2 ± 0.2 24.8 ± 0.1 25.1 ± 0.3 0.9 [0.7-1.5] 0.7[0.3-2.1] 1.0 [0.5-1.3] iNOS 36.2 ± 0.5 35.0 ± 0.2 34.8 ± 0.8 1.1 ± 0.31.8 ± 0.5 2.6 ± 0.8 Adenosine receptors A1 28.6 ± 0.3 27.9 ± 0.3 29.3 ±0.6 1.3 [0.4-2.3] 0.7 [0.5-1.4] 0.8 [0.3-1.5] A2A 27.0 ± 0.1 26.4 ± 0.227.3 ± 0.3 1.3 ± 0.4 0.8 ± 0.2 0.9 ± 0.2 A2B 29.0 [28.9-29.3] 28.4[28.1-29.0] 29.0 [28.9-30.2] 1.2 ± 0.3 0.8 ± 0.2 0.8 ± 0.1 A3 34.3 ± 0.334.2 ± 0.4 35.0 ± 0.6 1.0 ± 0.1  0.5 ± 0.1^(#) 0.7 ± 0.1 Data isexpressed as mean ± SEM, and median [25^(th) percentile, 75^(th)percentile], depending on the distribution of each parameter.Significant differences of the fold increase was estimated usingKruskal-Wallis test with Dunns post-test or one-way ANOVA withBonferroni post-test. Placebo, LPS n = 6; LPS + recAP n = 5. ^(#)p <0.05 compared to placebo. * p < 0.05 compared to LPS. LPS,Lipopolysaccharide; recAP, recombinant Alkaline Phosphatase; MPO,myeloperoxidase; BAX, Bcl2-associated X protein; iNOS, inducible nitricoxide synthase.

Literature References to Example 9

-   Bauerle, J. D., Grenz, A., Kim, J. H., Lee, H. T., and    Eltzschig, H. K. 2011. Adenosine generation and signaling during    acute kidney injury. J Am Soc Nephrol 22:14-20.-   Chen, K. T., Malo, M. S., Moss, A. K., Zeller, S., Johnson, P.,    Ebrahimi, F., Mostafa, G., Alam, S. N., Ramasamy, S., Warren, H. S.,    et al. 2010. Identification of specific targets for the gut mucosal    defense factor intestinal alkaline phosphatase. Am J Physiol    Gastrointest Liver Physiol 299:G467-475.-   Di Sole, F. 2008. Adenosine and renal tubular function. Curr Opin    Nephrol Hypertens 17:399-407.-   Eltzschig, H. K., Sitkovsky, M. V., and Robson, S. C. 2012.    Purinergic signaling during inflammation. N Engl J Med    367:2322-2333.-   Kiffer-Moreira, T., Sheen, C. R., Gasque, K. C., Bolean, M.,    Ciancaglini, P., van Elsas, A., Hoylaerts, M. F., and    Milan, J. L. 2014. Catalytic signature of a heat-stable, chimeric    human alkaline phosphatase with therapeutic potential. PLoS One    9:e89374.-   Peters, E., Heemskerk, S., Masereeuw, R., and Pickkers, P. 2014.    Alkaline Phosphatase: A Possible Treatment for Sepsis-Associated    Acute Kidney Injury in Critically Ill Patients. Am J Kidney Dis.    63:1038-48-   Schock-Kusch, D., Sadick, M., Henninger, N., Kraenzlin, B., Claus,    G., Kloetzer, H. M., Weiss, C., Pill, J., and Gretz, N. 2009.    Transcutaneous measurement of glomerular filtration rate using    FITC-sinistrin in rats. Nephrol Dial Transplant 24:2997-3001.-   Schock-Kusch, D., Xie, Q., Shulhevich, Y., Hesser, J., Stsepankou,    D., Sadick, M., Koenig, S., Hoecklin, F., Pill, J., and    Gretz, N. 2011. Transcutaneous assessment of renal function in    conscious rats with a device for measuring FITC-sinistrin    disappearance curves. Kidney Int 79:1254-1258.-   Wilmer, M. J., Saleem, M. A., Masereeuw, R., Ni, L., van der    Velden, T. J., Russel, F. G., Mathieson, P. W., Monnens, L. A., van    den Heuvel, L. P., and Levtchenko, E. N. 2010. Novel conditionally    immortalized human proximal tubule cell line expressing functional    influx and efflux transporters. Cell Tissue Res 339:449-457.

Example 10

Comparison LVL-RecAP with RecAP under different temperature conditions

10.6. Materials 10.6.1 Reference Standards LVL-RecAP

-   -   Batch number: NB1963p1    -   PRA ID: 14-049    -   Protein content: 9.9 mg/mL (OD280)    -   Activity: 6537 U/mL (660 U/mg)    -   Storage condition: nominal at −70° C.    -   Expiry date: 8 Jan. 2015

RecAP

-   -   Batch number: 2013-052, lot 62    -   PRA ID: 14-311    -   Protein content: 13.3 mg/mL (OD280)    -   Activity: 9871 U/mL (742 U/mg)    -   Storage condition: at 2-8° C.    -   Expiry date: 6 Jun. 2016

10.6.2 Blank Matrix

The following biological matrix was used for preparation of samplesolutions.

-   -   Matrix: serum    -   Species: human    -   Supplier: Sera Laboratories International, Haywards Heath, UK    -   Storage condition: At a nominal storage temperature −20° C.    -   PRA IDs: 14-0624, 14-0647 and 14-0652    -   Expiry dates: 2 May 2016 (14-0624),        -   6 May 2016 (14-0647 and 14-0652)

10.7. Methods 10.7.1 Preparation of Solutions 10.7.1.1 2 M SodiumHydroxide

A 2 molar sodium hydroxide solution was prepared by dissolving 8 g ofsodium hydroxide in approximately 90 mL Milli-Q water and after coolingto room temperature the volume was adjusted to 100 mL. The solution wasstored at room temperature up to a maximum of one month.

10.7.1.2 1 M Magnesium Chloride

A 1 molar magnesium chloride solution was prepared by dissolving 4.06 gmagnesium chloride hexahydrate in approximately 16 mL Milli-Q water.After dissolving the volume was adjusted to 20 mL. The solution wasstored at nominal +4° C. up to a maximum of one month.

10.7.1.3 0.1 M Zinc Chloride

A 0.1 molar zinc chloride solution was prepared by dissolving 272.5 mgzinc chloride in approximately 16 mL Milli-Q water. After dissolving,the volume was adjusted to 20 mL.

The solution was stored at nominal +4° C. up to a maximum of one month.

10.7.1.4 0.025M Glycine pH 9.6 Solution for 25° C. Method

A 0.025 molar glycine pH 9.6 solution was prepared by dissolving 3.76 gof glycine in approximately 1800 mL Milli-Q water. The solution waswarmed to 25° C. and adjusted to pH 9.6 with 2 M sodium hydroxide (seeSection 10.7.1.1). The volume was made up to 2000 mL and the pH wasrechecked. The pH should be pH 9.6 at 25° C. The solution was stored atnominal +4° C. up to a maximum of one week.

10.7.1.5 Enzyme Diluent Buffer for 25° C. Method

The enzyme diluent buffer was prepared by mixing 0.5 mL 1 M magnesiumchloride (see Section 10.7.1.2) with 0.5 mL 0.1 M zinc chloride (seeSection 10.7.1.3) and 500 mL 0.025 M glycine pH 9.6 solution (seeSection 10.7.1.4). To this solution, 5.00 g mannitol and 0.25 g bovineserum albumin was added and dissolved under stirring. The pH was checkedand if deemed necessary adjusted to pH 9.6 at 25° C. using 2 M sodiumhydroxide. The enzyme diluent buffer was prepared freshly every day.

10.7.1.6 0.0103 M p-Nitrophenyl Phosphate pH 9.6 for 25° C. Method

A 0.0103 M p-nitrophenyl phosphate pH 9.6 was prepared by dissolving1528 mg p-nitrophenyl phosphate in approximately 360 mL 0.025 M glycinepH 9.6 solution (see Section 10.7.1.4). The pH was checked and if deemednecessary adjusted to pH 9.6 at 25° C. using 2 M sodium hydroxide (seeSection 10.7.1.1). After checking the pH, the volume was adjusted to 400mL with 0.025 M glycine pH 9.6 solution. The solution was stored atnominal +4° C. up to a maximum of 5 days.

10.7.1.7 Working Substrate for 25° C. Method

Working substrate was prepared by mixing 120 mL 0.0103 M p-nitrophenylphosphate pH 9.6 solution (see Section 10.7.1.6) with 1.25 mL 1 Mmagnesium chloride solution (see Section 10.7.1.2). To this solutionapproximately 15 mL 0.025 M glycine pH 9.6 solution (see Section10.7.1.4) was added and the pH was checked and if deemed necessaryadjusted to pH 9.6 at 25° C. using 2 M sodium hydroxide (see Section10.7.1.1). The volume was adjusted to 145 mL with 0.025 M glycine pH 9.6solution. Working substrate was prepared freshly every day.

10.7.1.8 0.025M Glycine pH 9.6 Solution for 37° C. Method

A 0.025 molar glycine pH 9.6 solution was prepared by dissolving 3.76 gof glycine in approximately 1800 mL Milli-Q water. The solution waswarmed to 25° C. and adjusted to pH 9.6 with 2 M sodium hydroxide (seeSection 10.7.1.1). The volume was made up to 2000 mL and the pH wasrechecked. The pH should be pH 9.6 at 37° C. The solution was stored atnominal +4° C. up to a maximum of one week.

10.7.1.9 Enzyme Diluent Buffer for 37° C. Method

The enzyme diluent buffer was prepared by mixing 0.5 mL 1 M magnesiumchloride (see Section 10.7.1.2) with 0.5 mL 0.1 M zinc chloride (seeSection 10.7.1.3) and 500 mL 0.025 M glycine pH 9.6 solution (seeSection 10.7.1.8). To this solution, 5.00 g mannitol and 0.25 g bovineserum albumin was added and dissolved under stirring. The pH was checkedand if deemed necessary adjusted to pH 9.6 at 37° C. using 2 M sodiumhydroxide. The enzyme diluent buffer was prepared freshly every day.

10.7.1.10 0.0103 M p-Nitrophenyl Phosphate pH 9.6 for 37° C. Method

A 0.0103 M p-nitrophenyl phosphate pH 9.6 was prepared by dissolving1528 mg p-nitrophenyl phosphate in approximately 360 mL 0.025 M glycinepH 9.6 solution (see Section 10.7.1.8). The pH was checked and if deemednecessary adjusted to pH 9.6 at 37° C. using 2 M sodium hydroxide (seeSection 10.7.1.1). After checking the pH, the volume was adjusted to 400mL with 0.025 M glycine pH 9.6 solution. The solution was stored atnominal +4° C. up to a maximum of 5 days.

10.7.1.11 Working Substrate for 37° C. Method

Working substrate was prepared by mixing 120 mL 0.0103 M p-nitrophenylphosphate pH 9.6 solution (see Section 10.7.1.10) with 1.25 mL 1 Mmagnesium chloride solution (see Section 10.7.1.2). To this solutionapproximately 15 mL 0.025 M glycine pH 9.6 solution (see Section10.7.1.4) was added and the pH was checked and if deemed necessaryadjusted to pH 9.6 at 37° C. using 2 M sodium hydroxide (see Section10.7.1.1). The volume was adjusted to 145 mL with 0.025 M glycine pH 9.6solution. Working substrate was prepared freshly every day.

10.7.2 LVL-RecAP Spike Solution (500 μg/mL)

A recAP spike solution were prepared by diluting 252.5 μL LVL-RecAP(Section 10.6.1) to 5.00 mL with enzyme diluent buffer (Section10.7.1.5). The final concentration of the spike solution is 500 μg/mL,this solution was used for preparation of the recAP spiked human serumsamples (Section 10.7.4).

10.7.3 RecAP Spike Solution (500 μg/mL)

A RecAP spike solution was prepared by diluting 188.0 μL RecAP (Section10.6.1) to 5.00 mL with enzyme diluent buffer (Section 10.7.1.5). Thefinal concentration of the spike solution is 500 μg/mL, this solutionwas used for preparation of the RecAP spiked human serum samples(Section 10.7.5).

10.7.4 Preparation of LVL-recAP Spiked Human Serum Samples

Serum samples were prepared by spiking LVL-RecAP to three individualblank serum batches using the following concentration:

LVL-RecAP Calculated Spike Total serum Concentration activity volumevolume samples (μg/mL) (U/L) (μL) (mL) 1 10.0 6603 100 5.00 2 8.00 528280.0 5.00 3 6.00 3962 60.0 5.00 4 4.00 2641 40.0 5.00 5 2.00 1321 20.05.00 6 1.00 660 10.0 5.00 7 endogenous endogenous 0 5.00 The serumsamples were stored at nominal −70° C. until analysis.

10.7.5 Preparation of RecAP Spiked Human Serum Samples

Serum samples were prepared by spiking RecAP to three individual blankserum batches using the following concentration:

RecAP Calculated Spike Total serum Concentration activity volume volumesamples (μg/mL) (U/L) (μL) (mL) 1 9.00 6680 90.0 5.00 2 7.20 5344 72.05.00 3 5.40 4088 54.0 5.00 4 3.60 2672 36.0 5.00 5 1.80 1336 18.0 5.00 60.900 668 9.00 5.00 7 endogenous endogenous 0 5.00 The serum sampleswere stored at nominal −70° C. until analysis.

10.7.6 Preparation of Sample Solutions for LVL-RecAP and RecAP EnzymeActivity

The sample solutions for LVL-RecAP and RecAP enzyme activity wereprepared by dilution of the LVL-RecAP product sample or RecAP productsample using enzyme diluent buffer (Section 10.7.1.5 for 25° C. methodor Section 10.7.1.9 for 37° C. method).

Sample 1 from LVL-RecAP and/or RecAP sample was diluted by taking 125 μLof the LVL-RecAP and/or RecAP serum sample and diluted to 2.50 mL withenzyme diluent buffer to prepare the final sample solution for LVL-RecAPor RecAP enzyme activity.

Sample 2 from LVL-RecAP and/or RecAP sample was diluted by taking 125 μLof the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL withenzyme diluent buffer to prepare the final sample solution for LVL-RecAPor RecAP enzyme activity.

Sample 3 from LVL-RecAP and/or RecAP sample was diluted by taking 167 μLof the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL withenzyme diluent buffer to prepare the final sample solution for LVL-RecAPor RecAP enzyme activity.

Sample 4 from LVL-RecAP and/or RecAP sample was diluted by taking 250 μLof the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL withenzyme diluent buffer to prepare the final sample solution for LVL-RecAPor RecAP enzyme activity.

Sample 5 from LVL-RecAP and/or RecAP sample was diluted by taking 500 μLof the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL withenzyme diluent buffer to prepare the final sample solution for LVL-RecAPand/or RecAP enzyme activity.

Sample 6 from LVL-RecAP and/or RecAP sample was diluted by taking 1000μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mLwith enzyme diluent buffer to prepare the final sample solution forLVL-RecAP and/or RecAP enzyme activity.

The endogenous (sample 7) from LVL-RecAP and/or RecAP sample was dilutedby taking 1000 μL of the LVL-RecAP and/or RecAP serum sample and dilutedto 2.00 mL with enzyme diluent buffer to prepare the final samplesolution for LVL-RecAP and/or RecAP enzyme activity.

10.8. Execution, Results and Discussion 10.8.1 Equipment and Settings

The following method and settings were used:

Spectrophotometer: Thermo Fisher Evolution 300 UV/VIS with single cellPeltier heater set at 25° C. (AN-18-3) or 37° C. (AN-18-4) with magneticstirrer

Wavelength: 405 nm

Measurement: For 3 minutes, each 15 seconds a measurement. The firstminute was not taken into account for the calculations.Cuvette type: Glass

The following solutions were pipetted into a glass cuvette. Thetemperature of the solutions was 25° C.±0.5° C. for AN-18-3 or 37°C.±0.5° C. for AN-18-4.

Reagent Sample solution Blank Working substrate 1450 μL 1450 μL Enzymediluent 50.0 μL Sample 50.0 μL Total volume 1500 μL 1500 μL

First the working substrate and the enzyme diluent were mixed before thesample was added. The solution was mixed and the cuvette was placedimmediately in the spectrophotometer and the increase in absorbance wasmeasured from 1 to 3 minutes in steps of 15 seconds to obtain at least 9data points. At the 3 minute (last) data point the Optical density was≤1.5. Furthermore linearity was acceptable; the correlation coefficient(r) for each replicate should be ≥0.990. All sample results with acorrelation coefficient (r) ≥0.990 were taken into account for theevaluation, sample results with a correlation coefficient (r)<0.990 werereported for information only. The enzyme activity was performed induplicate mode, one test of each dilution.

10.8.2 LVL-RecAP and RecAP Enzyme Activity

Although in the Study Plan was described that the difference for the twoindividual results (enzyme activity) should be ≤5.0% to accept the twoindividual dilution results, all results with a correlation coefficient(r) for each measurement ≥0.990 were used for evaluation. This becausethe enzyme activity method was validated for LVL-RecAP drug productsamples at 25° C.±0.5° C. and not for other Alkaline Phosphatase originand/or other conditions.

FIG. 19 shows the correlation between enzyme activities of RecAP inhuman serum at 25° C. and at 37° C. FIG. 20 shows the correlationbetween enzyme activities of LVL-RecAP in human serum at 25° C. and at37° C.

FIG. 20 shows the activity/μg protein at 25° C. and at 37° C. for RecAPand LVL-RecAP. Values represent mean Δactivity between 25° C. and 37° C.for a given protein concentration.

1-4. (canceled)
 5. A method for treating a subject suffering from or atrisk of suffering from a disease accompanied by a local or systemic zincdeficiency comprising administering to the subject: (1) a protein havingphosphatase activity, (2) a polynucleotide comprising a nucleic acidsequence encoding said protein, or (3) a vector comprising saidpolynucleotide, wherein said protein comprises an amino acid sequence ofat least 200 consecutive amino acids having at least 90% sequenceidentity with SEQ ID NO: 5, an amino acid sequence of at least 50consecutive amino acids having at least 90% sequence identity with SEQID NO: 6, and an amino acid sequence of at least 40 consecutive aminoacids having at least 90% sequence identity with SEQ ID NO: 7, whereinthe full length protein comprises an amino acid sequence having at least90% sequence identity with the full length amino acid sequence of SEQ IDNO: 1, with the proviso that the amino acid at position 279 is leucine(L), the amino acid at position 328 is valine (V) and the amino acid atposition 478 is leucine (L).
 6. A method for treating a subjectsuffering from or at risk of suffering from an inflammatory disease, akidney disease or hypophosphatasia comprising administering to thesubject: (1) a protein having phosphatase activity, (2) a polynucleotidecomprising a nucleic acid sequence encoding said protein, or (3) avector comprising said polynucleotide, wherein said protein comprises anamino acid sequence of at least 200 consecutive amino acids having atleast 90% sequence identity with SEQ ID NO: 5, an amino acid sequence ofat least 50 consecutive amino acids having at least 90% sequenceidentity with SEQ ID NO: 6, and an amino acid sequence of at least 40consecutive amino acids having at least 90% sequence identity with SEQID NO: 7, wherein the full length protein comprises an amino acidsequence having at least 90% sequence identity with the full lengthamino acid sequence of SEQ ID NO: 1, with the proviso that the aminoacid at position 279 is leucine (L), the amino acid at position 328 isvaline (V) and the amino acid at position 478 is leucine (L).
 7. Themethod according to claim 6, wherein said inflammatory disease isselected from the group consisting of autoimmune diseases, rheumatoidarthritis, asthma, chronic obstructive pulmonary disease,atherosclerosis, inflammatory disease of the gastro-intestinal tract,infection, sepsis, neurodermatitis, inflammatory liver disease,inflammatory lung disease, and inflammatory kidney disease.
 8. Themethod according to claim 6, wherein said kidney disease is selectedfrom the group consisting of kidney injury, acute kidney injury, chronickidney disease, renal failure, acute renal failure, ischemic renaldisease and ischemia/reperfusion kidney damage.
 9. The method accordingto claim 6, wherein said hypophosphatasia is selected from the groupconsisting of perinatal hypophosphatasia, infantile hypophosphatasia,childhood hypophosphatasia, and adult hypophosphatasia. 10-15.(canceled)
 16. The method according to claim 6, wherein said treatmentof said subject suffering from or at risk of suffering fromhypophosphatasia results in prolonged survival, increased body weight,improved skeletal phenotype, attenuation of craniofacial defects,improvement of dento-alveolar phenotype, and/or reduction of plasma PPilevels.
 17. The method according to claim 5, wherein the protein havingphosphatase activity comprises an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 1, withthe proviso that the amino acid at position 279 is leucine (L), theamino acid at position 328 is valine (V) and the amino acid at position478 is leucine (L).
 18. The method according to claim 5, wherein theprotein having phosphatase activity comprises an amino acid sequencehaving at least 98% sequence identity to the amino acid sequence of SEQID NO: 1, with the proviso that the amino acid at position 279 isleucine (L), the amino acid at position 328 is valine (V) and the aminoacid at position 478 is leucine (L).
 19. The method according to claim5, wherein the protein having phosphatase activity comprises an aminoacid sequence of 300-365 consecutive amino acids having at least 95%sequence identity with SEQ ID NO: 5, an amino acid sequence of 60-65consecutive amino acids having at least 95% sequence identity with SEQID NO: 6, and an amino acid sequence of 50-54 consecutive amino acidshaving at least 95% sequence identity with SEQ ID NO: 7, wherein thefull length protein comprises an amino acid sequence having at least 95%sequence identity with the full length amino acid sequence of SEQ ID NO:1, with the proviso that the amino acid at position 279 is leucine (L),the amino acid at position 328 is valine (V) and the amino acid atposition 478 is leucine (L).
 20. The method according to claim 5,wherein the protein having phosphatase activity comprises an amino acidsequence of 350-365 consecutive amino acids having at least 98% sequenceidentity with SEQ ID NO: 5, an amino acid sequence of 62-65 consecutiveamino acids having at least 98% sequence identity with SEQ ID NO: 6, andan amino acid sequence of 52-54 consecutive amino acids having at least98% sequence identity with SEQ ID NO: 7, wherein the full length proteincomprises an amino acid sequence having at least 98% sequence identitywith the full length amino acid sequence of SEQ ID NO: 1, with theproviso that the amino acid at position 279 is leucine (L), the aminoacid at position 328 is valine (V) and the amino acid at position 478 isleucine (L).
 21. The method according to claim 5, wherein the proteinhaving phosphatase activity comprises the amino acid sequence as setforth in SEQ ID NO:
 1. 22. The method according to claim 5, wherein theprotein having phosphatase activity consists of the amino acid sequenceas set forth in SEQ ID NO:
 1. 23. The method according to claim 6,wherein the protein having phosphatase activity comprises an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 1, with the proviso that the amino acid atposition 279 is leucine (L), the amino acid at position 328 is valine(V) and the amino acid at position 478 is leucine (L).
 24. The methodaccording to claim 6, wherein the protein having phosphatase activitycomprises an amino acid sequence having at least 98% sequence identityto the amino acid sequence of SEQ ID NO: 1, with the proviso that theamino acid at position 279 is leucine (L), the amino acid at position328 is valine (V) and the amino acid at position 478 is leucine (L). 25.The method according to claim 6, wherein the protein having phosphataseactivity comprises an amino acid sequence of 300-365 consecutive aminoacids having at least 95% sequence identity with SEQ ID NO: 5, an aminoacid sequence of 60-65 consecutive amino acids having at least 95%sequence identity with SEQ ID NO: 6, and an amino acid sequence of 50-54consecutive amino acids having at least 95% sequence identity with SEQID NO: 7, wherein the full length protein comprises an amino acidsequence having at least 95% sequence identity with the full lengthamino acid sequence of SEQ ID NO: 1, with the proviso that the aminoacid at position 279 is leucine (L), the amino acid at position 328 isvaline (V) and the amino acid at position 478 is leucine (L).
 26. Themethod according to claim 6, wherein the protein having phosphataseactivity comprises an amino acid sequence of 350-365 consecutive aminoacids having at least 98% sequence identity with SEQ ID NO: 5, an aminoacid sequence of 62-65 consecutive amino acids having at least 98%sequence identity with SEQ ID NO: 6, and an amino acid sequence of 52-54consecutive amino acids having at least 98% sequence identity with SEQID NO: 7, wherein the full length protein comprises an amino acidsequence having at least 98% sequence identity with the full lengthamino acid sequence of SEQ ID NO: 1, with the proviso that the aminoacid at position 279 is leucine (L), the amino acid at position 328 isvaline (V) and the amino acid at position 478 is leucine (L).
 27. Themethod according to claim 5, wherein the protein having phosphataseactivity comprises the amino acid sequence as set forth in SEQ ID NO: 1.28. The method according to claim 6, wherein the protein havingphosphatase activity consists of the amino acid sequence as set forth inSEQ ID NO: 1.